Treatment for nicotine-induced lung disease using peroxisome proliferator-activated receptor gamma agonists

ABSTRACT

This invention pertains to the discovery that nicotine interrupts molecular signaling between endodermal and mesodermal cells of the lung alveolus. Treatment of the lung with specific molecular agents (e.g., PPAR gamma agonists) can prevent and/or reverse the injury caused by nicotine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/296,656, filed Oct. 9, 2008, which was filed as a national stageapplication under 35 U.S.C. §371 of PCT International Application No.PCT/US07/08751, filed Apr. 10, 2007, which claims benefit of andpriority to U.S. Patent Application Ser. No. 60/791,612, filed on Apr.11, 2006, and U.S. Patent Application Ser. No. 60/813,430, filed on Jun.13, 2006. Each of the foregoing applications are incorporated herein byreference in their entirety for all purposes.

FIELD OF THE INVENTION

This invention pertains to the area of lung diseases. In certainembodiments, this invention provides methods and compositions to treator prevent nicotine-induced lung damage.

BACKGROUND OF THE INVENTION

Maternal smoking during pregnancy has diverse adverse effects on fetaloutcome, including increased risk of spontaneous abortion, stillbirth,premature delivery, low birth weight, early neonatal mortality, suddeninfant death syndrome, and poor pulmonary outcome (Cunningham et al.(1994) Am. J. Epidemiol. 139: 1139-1152; Gilliland et al. (2003) Am. J.Respir. Crit. Care Med. 167: 917-924.; Hanrahan et al. (1992) Am. Rev.Respir. Dis. 145: 1129-1135; Higgins (2002) Curr Opin Obstet Gynecol 14:145-151; Hofhuis et al. (2003) Arch. Dis. Child 88: 1086-1090.).Maternal smoking during pregnancy adversely affects fetal lung growththat may result in adverse long-term consequences such as increasedoccurrence of lower respiratory illnesses and altered pulmonarymechanics on pulmonary function testing (Chen et al. (1987) Pediatr.Pulmonol., 3: 51-58; Collins et al. (1985) Pediatr. Res. 19: 408-412;Gilliland et al. (2003) Am. J. Respir. Crit. Care Med. 167: 917-924;Hofhuis et al. (2003) Arch. Dis. Child., 88: 1086-1090; Maritz (1988)Biol. Neonate, 53: 163-170; Scott (2004) Tobacco Induced Diseases 2:3-25; Walsh (1994) Hum. Biol., 66: 1059-1092). The mechanisms underlyingthe general effects of maternal smoking on fetal viability and growthare generally thought to be due to fetal hypoxia (Cnattingius andNordstrom (1996) Acta Paediatr. 85: 1400-1402). The mechanismsunderlying pulmonary outcomes, however, appear to be more complex andare poorly understood (Scott (2004) Tobacco Induced Diseases 2: 3-25).On the one hand, there is evidence of increased surfactant production atbirth, possibly contributing to a decrease in the incidence ofrespiratory distress syndrome (Curet et al. (1983) Am. J. Obstet.Gynecol. 147: 446-450; Gluck and Kulovich (1973) Am J Obstet Gynecol.115: 539-546; Lieberman et al. (1992) Obstet. Gynecol. 79: 564-570; 39.Wuenschell et al. (1998) Am. J. Physiol. Lung Cell Mol. Physiol. 274:L165-L170). On the other hand, there is strong evidence for deleteriouseffects on pre- and postnatal lung growth and development following inutero exposure to maternal smoking (Chen et al. (1987) Pediatr Pulmonol3: 51-58; Cnattingius and Nordstrom (1996) Acta Paediatr 85: 1400-1402;Collins et al. (1985) Pediatr. Res., 19: 408-412; Cunningham et al.(1994) Am J Epidemiol 139: 1139-1152; Gilliland et al. (2003) Am. J.Respir. Crit. Care Med., 167: 917-924; Hanrahan et al. (1992) Am. Rev.Respir. Dis., 145: 1129-1135; Higgins (2002) Curr. Opin. Obstet.Gynecol., 14: 145-151; Hofhuis et al. (2003) Arch Dis Child 88:1086-1090; Maritz (1988) Biol Neonate 53: 163-170; Scott (2004) TobaccoInduced Diseases 2: 3-25; Sekhon et al. (1999) J Clin Invest 103:637-647; Sekhon et al. (2001) Am. J. Respir. Crit. Care Med., 164:989-994; Walsh (1994) Hum. Biol., 66: 1059-1092). The mechanismsunderlying these seemingly paradoxical effects remain largely unknown.

There is, consequently, currently no specific treatment for thedeleterious effects of smoking on the lung. Such patients are typicallytreated with steroids and β blockers, which alleviate the symptomscaused by smoking, but do not address the actual etiology of thesmoke-induced effects.

SUMMARY OF THE INVENTION

This invention pertains to the elucidation of a mechanism of alteredprenatal and postnatal lung development and function and provides amolecular approach to its prevention.

Both normal lung development and injury/repair utilize commonmesenchymal-epithelial signaling pathways to maintain homeostasis.Epithelially-derived parathyroid hormone-related protein (PTHrP) inducesthe differentiation of mesodermal alveolar interstitial fibroblasts tolipid-containing interstitial lipofibroblasts (LIF) via a PTHrPreceptor-mediated, cAMP-dependent PKA pathway. Other important keyproteins in this pathway include peroxisome proliferator-activatedreceptor (PPAR) and adipocyte differentiation-related protein (ADRP).The lipid-containing LIFs produce factors that induce the growth anddifferentiation of the adjoining type II cells, culminating in alveolarhomeostasis. Factors that disrupt this cellular homeostatic mechanism bycausing the transdifferentiation of LIFs to myofibroblasts (MYFs) leadto abnormal lung development and function. Using embryonic WI38 humanlung fibroblasts as a model, we tested the hypothesis that in vitronicotine exposure specifically disrupts PTHrP-mediated alveolarepithelial-mesenchymal paracrine signaling that results in alveolarLIF-to-MYF transdifferentiation, resulting in altered pulmonary growthand differentiation. Furthermore, demonstrate that by targeting thespecific molecular elements that maintain the LIF phenotype,nicotine-induced LIF-to-MYF such transdifferentiation could beprevented.

Thus, in certain embodiments, this invention provides methods ofreducing, eliminating, and/or reversing nicotine damage in a mammal. Themethods typically involve contacting pulmonary tissue in the mammal witha PTHrP signaling agonist. The PTHrP signaling agonist is typicallyprovided as sufficient dosage to reduce, eliminate, and/or reversenicotine damage in the mammal. In certain embodiments the PTHrPsignaling agonist comprises a PPAR gamma (PPARγ) agonist. In certainembodiments the mammal is a pregnant mammal and the pulmonary tissue isin a developing fetus. In certain embodiments the mammal is a pregnantmammal and said pulmonary tissue comprises pulmonary tissue of saidpregnant mammal. In certain embodiments the mammal is a human smoker. Incertain embodiments the mammal is a non-human mammal or a human, exposedto second-hand smoke. In certain embodiments the human is a humandiagnosed as having asthma, chronic obstructive pulmonary disease, lungcancer, or emphysema. In certain embodiments the human is a human thatis formerly a smoker. In various embodiments the PPAR gamma agonist is athiozolidinedione. In certain embodiments PPAR gamma agonist includesone or more agents independently selected from the group consisting ofrosiglitazone, troglitazone (Resulin), farglitazar, phenylacetic acid,GW590735, GW677954, Avandia, Avandamet (avandia+metformin), ciglitazone,15 deoxy prostaglandin J2 (15PGJ2), pioglitazone (Actos),15-deoxy-delta12,14 PGD2, MCC-555, and triterpenoids (e.g.,2-Cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), CDDO-Me, and CDDOIm(see, e.g., Chintharlapalli, et al. (2005) Mol. Pharmacol., 68:119-128), and the like). In certain embodiments the PTHrP signalingagonist (e.g., PPAR gamma agonist) is administered via an inhalationroute. In certain embodiments the PTHrP signaling agonist (e.g., PPARgamma agonist) is administered via a nasal spray. In certain embodimentsthe PTHrP signaling agonist (e.g., PPAR gamma agonist) is administeredvia an oral inhaler. In certain embodiments the PTHrP signaling agonist(e.g., PPAR gamma agonist) is administered orally. In certainembodiments the PTHrP signaling agonist (e.g., PPAR gamma agonist) isadministered systemically.

Also provided are methods for inhibiting or repairing deleteriouseffects of smoking on the lung. The methods typically involve methodcomprising administering to a subject in need thereof a sufficientamount of a PTHrP signaling agonist (e.g., PPAR gamma agonist) toinhibit or repair smoking-induced damage to the lung.

Methods are provided for screening for an agent that inhibits or repairsa deleterious effect of smoking on the lung. The methods typicallyinvolve screening the test agent for PTHrP signaling agonistic activity(e.g., PPAR gamma agonist activity), where the PTHrP signaling activityis an indicator that the test agent is a candidate agent for thetreatment or prevention of smoking-induced lung damage.

Methods are also provided for screening for an agent that mitigates oneor more symptoms of nicotine-induced pulmonary damage. The methodstypically involve exposing a test agent to a test mammal or to amammalian cell; and determining the expression or activity of a PTHrPsignaling pathway component (e.g., PPAR gamma and/or a PPAR gammareceptor), where an increase in the component expression or activity inthe test mammal indicates that the test agent is a good candidate agentfor mitigating, stopping, or reversing one or more symptoms ofnicotine-induced pulmonary damage in a mammal. In certain embodimentsthe determining the expression comprises measuring the level of nucleicacid encoding PPAR gamma and/or a PPAR gamma receptor. In certainembodiments the measuring comprises measuring the level of expressedPPAR gamma and/or PPAR gamma receptor protein. In various embodimentsthe measuring is via a method selected from the group consisting ofcapillary electrophoresis, a Western blot, mass spectroscopy, ELISA,immunochromatography, and immunohistochemistry. In various embodimentsthe determining the expression comprises measuring the level of mRNAencoding PPAR gamma and/or a PPAR gamma receptor. In certain embodimentsthe level of PPAR gamma mRNA or PPAR gamma receptor mRNA is measured byhybridizing the mRNA to a probe that specifically hybridizes to a PPARgamma or PPAR gamma receptor nucleic acid (e.g., via a method selectedfrom the group consisting of a Northern blot, a Southern blot using DNAderived from the PPAR gamma RNA and/or PPAR gamma receptor RNA, an arrayhybridization, an affinity chromatography, and an in situhybridization). In certain embodiments the level of PPAR gamma and/orPPAR gamma receptor mRNA is measured using a nucleic acid amplificationreaction.

In various embodiments kits are provided for mitigating or reversingnicotine-induce pulmonary damage. The kits typically comprises acontainer containing one or more PTHrP pathway signaling agonists (e.g.,PPAR gamma agonists) and instructional materials teaching the use ofPPAR gamma agonists in the treatment of nicotine-induced pulmonarydamage.

Also provided is the use of a PTHrP signaling agonist (e.g., PPAR gammaagonist) in the treatment of nicotine-induced pulmonary damage.

In certain embodiments, use of a PTHrP signaling agonist (e.g., PPARgamma agonist) in the manufacture of a medicament for the treatment ofnicotine-induced pulmonary damage is also provided.

In certain embodiments of the methods and treatments described herein,the mammal is not a human or non-human mammal diagnosed with and/orbeing treated for asthma, chronic obstructive pulmonary disease, lungcancer, or emphysema and/or is not being treated for diabetes, and/orobesity or anexoria, and/or an eating or appetite disorder.

DEFINITIONS

The terms “PPAR gamma ligand” and “PPAR gamma agonist” are usedinterchangeably and refers to an agent that upregulates directly orindirectly activity of a pathway mediated by a PPAR gamma receptor.PPAR-γ agonists include agents that, when interacting directly orindirectly with PPAR-γ, increase the biological activity of PPAR-γ(e.g., the ability of PPAR-γ to inhibit Egr-1 expression).

The term “a PTHrP receptor” is used to mean a receptor that binds toPTHrP, and examples include a PTHrP type I receptor (described, e.g., inJapanese Patent Application Laying-Open (Kohyo) No. 6-506598).

The term “second-hand smoke” refers to smoke, typically smoke producedby burning tobacco or a burning tobacco product that is inhaled orotherwise contacted by a person other than the person who is utilizingthe burning tobacco or burning tobacco product (e.g., the smoker).

The term “administering” when used herein with respect to a PPARγagonist includes, but is not limited to giving, providing, feeding,dispensing, inserting, injecting, infusing, perfusing, prescribing,furnishing, treating with, taking, spraying, inhaling, swallowing,eating, or applying a pharmaceutically acceptable PPARγagonist-containing composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the expression of nicotine acetylcholine (nACh)receptors 3 and 7 by control and nicotine-stimulated WI38 cells byRT-PCR (FIG. 1A) and Western blot analysis (FIG. 1B), respectively. Onnicotine (1×10⁻⁹ or 1×10⁻⁶ M) stimulation for 7 days, there weresignificant increases in the expressions of both nACh receptors α₃ andα₇ (*P<0.05 vs. control by ANOVA, n=3).

FIG. 2 shows that nicotine treatment of cultured WI38 cells for 7 daysresults in significant decreases in parathyroid hormone-related protein(PTHrP) receptor, peroxisome proliferator-activated receptor γ (PPARγ),and adipocyte differentiation-related protein (ADRP), and a significantincrease in α-smooth muscle actin (α-SMA) mRNA expression (*P<0.05 vs.control by ANOVA, n=3). Representative RT-PCR blots (top) anddensitometric histograms (bottom) for PTHrP receptor, PPAR, ADRP, and-SMA mRNA expression are shown.

FIG. 3 shows that nicotine treatment of cultured WI38 cells for 7 daysresults in significant decreases in PTHrP receptor, PPAR, and ADRP, anda significant increase in -SMA protein expression (*P<0.05 vs. controlby ANOVA, n=3). Representative Western blots (top) and densitometrichistograms (bottom) for PTHrP receptor, PPAR, ADRP, and α-SMA mRNAexpression are shown.

FIG. 4 shows representative immunofluorescence staining for lipiddroplets (red staining) and α-SMA (green staining) in cultured WI38cells with and without nicotine (1 ×10⁻⁹ M) treatment for 7 days isshown. Cultured WI38 cells were stained for lipid droplets alone usingoil red O, α-SMA using specific monoclonal antibody, and nuclei using4′,6′-diamidino-2-phenylindole (DAPI). Bottom: shows triple stainingusing oil red O, α-SMA-specific antibody, and DAPI. Nicotine treatmentmarkedly reduced staining for lipid droplets and markedly increasedα-SMA expression. This can be clearly seen in the bottom panel thatshows cytoplasmic colocalization of staining for lipid droplets andα-SMA.

FIG. 5 shows that nicotine treatment (1×10⁻⁹ M for 7 days) caused analmost 50% decrease in triglyceride uptake (*P<0.05 vs. control byANOVA, n=6), which was completely prevented by concomitant treatment ofWI38 cells with rosiglitazone (RGZ; 1×10⁻⁵ M), PTHrP (5×10⁻⁷ M), ordibutyryl cAMP (DBcAMP) (1×10⁻⁴ M).

FIGS. 6A-6C show that concomitant treatment with specific stimulants ofPTHrP-mediated, cAMP-dependent PKA lipogenic pathway, i.e., PTHrP(5×10⁻⁷ M), DBcAMP (1×10⁻⁴ M), or RGZ (1×10⁻⁵ M), completely preventsthe nicotine-induced decreases in PTHrP receptor (FIG. 6A) and PPARγ(FIG. 6B) protein, and increase in α-SMA (FIG. 6C) protein expressions(*P<0.05 vs. control by ANOVA, n=3), indicating prevention ofnicotine-induced lipofibroblast-to-myofibroblast transdifferentiation bythese agents.

FIG. 7 shows that transfection of WI38 cells with PPAR expression vectorcompletely prevented the nicotine-inducedlipofibroblast-to-myofibroblast transdifferentiation. WI38 cells weretransfected cells with either 4 or 8 μg of pCMX-PPAR cDNA. Then, thecells were treated with nicotine (1×10⁻⁹ or 1×10⁻⁶ M) for 7 days,following which the expressions of PPAR and -SMA were assessed byWestern blot analyses. Under control conditions (without transfection),there was a significant decrease in PPAR and a significant increase in-SMA protein expression. However, after PPARγ transfection, thesechanges were completely prevented. Representative blots out of 2independent experiments are shown.

FIG. 8 shows that nicotine (1×10⁻⁶ M) treatment caused a 30% decrease inPTHrP binding to its receptor (fmol·90 min⁻¹·mg protein⁻¹; *P<0.05 vs.control by ANOVA, n=6), which was completely prevented by pretreatmentwith either D-tubocurarine (1×10⁻⁶ M), a nonspecific nACh receptorantagonist, or α-bungarotoxin (1×10⁻⁶ M), an α₇ nACh receptorantagonist, but not by mecamylamine (1×10⁻⁹ or 1×10⁻⁶ M), an α₃ nAChreceptor antagonist.

FIG. 9 shows that nicotine treatment (1×10⁻⁹ M for 24 h) caused analmost 30% decrease in triglyceride uptake (*P<0.05 vs. control byANOVA, n=6), which was completely prevented by pretreatment with eitherD-tubocurarine (1×10⁻⁶ M), a nonspecific nACh receptor antagonist, orα-bungarotoxin (1×10⁻⁶ M), an α₇ nACh receptor antagonist, but not bymecamylamine (1×10⁻⁹ or 1×10⁻⁶ M), an 3 nACh receptor antagonist.

FIG. 10 schematically illustrates that nicotine adversely affectspulmonary alveolar epithelial-mesenchymal interactions, thereby inducinglipo-to-myofibroblast transdifferentiation.

FIG. 11 shows the effect of nicotine on expression of markers forlipo-to-myofibroblast differentiation. Time-mated pregnant SpragueDawley rat dams were treated with either placebo or nicotine (2 mg/kg)i. p. once daily from embryonic day (e) 6 of gestation until theirsacrifice on e20, following which mRNA expression for the markers oflipo-to-myofibroblast transdifferentiation in the whole lung tissue wasexamined. There was a significant decrease in PTHrP receptor, PPARγ, andADRP (*=p<0.05 vs controls) expression and a significant increase inαSMA mRNA expression

FIG. 12 shows the effect of PPARγ agonist on nicotine-induced alveolartype II cell proliferation. Following in utero nicotine administrationto the dam (1 mg/kg i.p. once daily from e6 to e20 gestation), there wasa 2-fold increase in alveolar type II cell proliferation vs. controlgroup. This increase was completely blocked by the concomitantadministration of a PPARγ agonist, PGJ₂ (p<0.05). A specific PPARγantagonist, GW9662, largely blocked the PGJ₂ effect on thenicotine-induced increase in alveolar type II cell proliferation to alarge extent (70%).

FIG. 13 shows surfactant phospholipid synthesis, as measured by[³H]choline incorporation into saturated phosphatidylcholine, bycultured alveolar type II cells, following in utero nicotine (1 mg/kgadministered i.p. once daily from e6 to e20 gestation to the pregnantmom) showed a significant increase in the nicotine-exposed group versusthe control group (p<0.05). The concomitant treatment, with either thePPARγ agonist PGJ₂, or the antagonist GW9662, had no effect on thenicotine-induced increase in phospholipid synthesis under in uteroconditions.

FIG. 14 shows that CTP: cholinephosphate cytidylyltransferase α (CCTα)protein expression by alveolar type II cells increased significantlyfollowing in utero (1 mg/kg administered i.p. once daily from e6 to e20gestation to the pregnant mom) (FIG. 1A) nicotine treatment. Theconcomitant treatment with either PGJ₂ or GW9662 had no effect on thenicotine-induced increase in CCTα protein expression under in uteroconditions

FIG. 15 shows that surfactant Protein-B expression increasedsignificantly following both in utero (1 mg/kg administered i.p. oncedaily from e6 to e20 gestation to the pregnant mom) nicotine treatment.Concomitant treatment with either PGJ₂ or GW9662 had no effect on thenicotine-induced increase in SP-B protein expression under in uteroconditions.

FIGS. 16A and 16B: Following in utero nicotine exposure (1 mg/kg i.p.administered once daily from e6 to e20 gestation to the pregnant mom),ribose synthesis, as measured by ¹³C glucose labeling, increasedsignificantly via the oxidative glucose-6-phosphate dehydrogenasepathway, while it decreased significantly via the non-oxidativetransketolase pathway (p<0.05; n=3 for both). The concomitantadministration of the PPARγ agonist PGJ₂ completely blocked thesechanges

FIGS. 17A and 17B: Following in utero nicotine exposure (1 mg/kg i.p.administered once daily from e6 to e20 gestation to the pregnant mom),de novo palmitate synthesis, as a function of total palmitate in thealveolar type II cells and the ¹³C carbon glucose labeling of theacetyl-CoA pool, increased significantly (p<0.05 for both). Theconcomitant administration of the PPARγ agonist PGJ₂ completely blockedthese changes.

FIGS. 18A-18F show the effect of nicotine on fetal alveolar type II(ATII) cell proliferation in vivo. In vivo fetal pulmonary ATII cellproliferation was determined immunohistochemically by double labelingwith a cell proliferation-specific marker, proliferating cell nuclearantigen (PCNA), and an ATII cell-specific marker, surfactant protein(SP)-C (FIGS. 18A and 18B), or assessed ex vivo by tetrazolium dye assay(FIG. 18C) after in utero nicotine administration to the dam (1 mg/kgip) once daily from embryonic day 6 to 20. Cell proliferation wasincreased ˜2-fold in the nicotine-exposed group vs. the control group.This increase was completely blocked by concomitant administration of aperoxisome proliferator-activated receptor (PPAR)-γ agonist, PGJ₂. Aspecific PPAR-γ antagonist, GW-9662, almost completely blocked the PGJ2effect on the nicotine-induced increase in ATII cell proliferation. Inlung explants in culture treated with nicotine for 24 h, similar to thein vivo data, there was a significant increase in ATII cellproliferation, which was blocked by PGJ₂; again, GW-9662 blocked thePGJ₂ effect (FIGS. 18D and 18E). Slides were examined at ×40magnification; black arrows in FIGS. 18A and 18D show ATII cells labeledwith PCNA (blue-gray nuclear stain) and SP-C (cytoplasmic stain). ATIIcells in 10 randomly selected areas (grid size 40,000 pm²) per slide (2slides/animal) were counted. FIG. 18F: no effect on cell proliferationin ATII cells directly stimulated in vitro with nicotine for 24 h.

FIGS. 19A-19B show the effect of nicotine on surfactantphosphatidylcholine synthesis in vivo and in vitro. Surfactantphospholipid synthesis measured by [³H]choline incorporation[disintegrations/min (dpm) per mg protein] into saturatedphosphatidylcholine by cultured ATII cells after in utero (1 mg/kg ipadministered once daily pregnant dam from embryonic day 6 to 20; FIG.19A) or in vitro (1×10⁻⁹ M for 24 h; FIG. 19B) nicotine treatment wassignificantly increased in the nicotine-exposed group vs. the controlgroup. Concomitant treatment with the PPAR-γ agonist PGJ₂ or antagonistGW9662 had no effect on the nicotine-induced increase in phospholipidsynthesis in utero or in vitro.

FIGS. 20A-20B, show the effect of nicotine on CTP:choline phosphatecytidylyl-transferase-α (CCT-α) protein expression in ATII cells. CCT-αprotein expression by ATII cells increased significantly after in utero(1 mg/kg ip administered to pregnant dam once daily from embryonic day 6to 20 (FIG. 20A), or in vitro (1×10⁻⁹ M for 24 h; FIG. 20B) nicotinetreatment. Concomitant treatment with PGJ₂ or GW9662 had no effect onthe nicotine-induced increase in CCT-α protein expression in utero or invitro. Representative Western blot and density histograms are shown.

FIGS. 21A-21B show the effect of nicotine on SP-B expression in ATIIcells. SP-B protein expression increased significantly after in utero (1mg/kg ip administered to pregnant dam once daily from embryonic day 6 to20; FIG. 21A) or in vitro (1×10⁻⁹ M for 24 h; FIG. 21B) nicotinetreatment. Concomitant treatment with PGJ2 or GW9662 had no effect onthe nicotine-induced increase in SP-B protein expression in utero or invitro. Representative Western blot and density histograms are shown.

FIGS. 22A-22D show the effect of in utero nicotine exposure on ATII cellmetabolism. After in utero nicotine exposure (1 mg/kg ip administered topregnant dam once daily from embryonic day 6 to 20), ribose synthesis,as measured by [¹³C]glucose labeling, increased significantly via theoxidative glucose-6-phosphate dehydrogenase (G6PD) pathway (FIG. 22A)and decreased significantly via the nonoxidative transketolase pathway(FIG. 22B). Concomitant administration of the PPAR-γ agonist PGJ₂completely blocked these changes. Direct in vitro treatment of culturedATII cells with nicotine did not alter ribose synthesis via theoxidative or nonoxidative pathway with or without PGJ2 (FIGS. 22C and22D).

FIGS. 23A-23D show the effect of nicotine on ATII cell de novo palmitatesynthesis. After in utero nicotine exposure (1 mg/kg ip administered topregnant dam once daily from embryonic day 6 to 20), de novo palmitatesynthesis, as a function of total palmitate in ATII cells (FIG. 23A) and[¹³C]glucose labeling of acetyl-CoA pool (FIG. 23B), increasedsignificantly. Concomitant administration of the PPAR-γ agonist PGJ₂completely blocked these changes. Direct in vitro treatment of culturedATII cells with nicotine did not alter de novo palmitate synthesis or[¹³C]glucose labeling of the acetyl-CoA pool with or without PGD2 (FIGS.23C and 23D).

FIG. 24 shows that parathyroid hormone-related protein, secreted by thealveolar type II (ATII) cell, binds to its receptor on thelipofibroblast, activating the cAMP-dependent PKA-mediated lipogenicpathway, upregulating PPARγ and its downstream target, ADRP, whichfacilitates triglyceride uptake by the lipofibroblast and leptin, whichstimulates surfactant phospholipid and protein synthesis by the alveolartype II cell. The triglycerides taken up by the lipofibroblast are thentrafficked to the ATII cell as substrate for surfactant phospholipidsynthesis. Nicotine exposure downregulates the PTHrP signaling pathway,resulting in LIF-to-MYF transdifferentiation. However, PTHrP signalingpathway agonists [PTHrP, dibutryl cAMP (DBcAMP), and rosiglitazone(RGZ)] can almost completely prevent nicotine-induced LIF-to-MYFtransdifferentiation

FIG. 25 shows that nicotine treatment of cultured lung fibroblasts for 7days results in significant decreases in parathyroid hormone-relatedprotein receptor (*p<0.001 vs. control; n=3), peroxisomeproliferator-activated receptor γ (*p<0.001 vs. control; n=3), andadipocyte differentiation-related protein (*p<0.05 vs. control; n=3),and significant increases in α smooth muscle actin (*p<0.001 vs.control; n=3) and calponin (*p<0.001 vs. control; n=3) proteinexpressions. The values are mean±SD. Representative Western blots anddensitometric histograms are shown

FIGS. 26A-26C: Embryonic human lung fibroblasts were initially exposedto nicotine (10⁻⁹M) for 7 days and then treated with PPARγ agonists[rosiglitazone (RGZ) (1×10⁻⁵ M), PTHrP (5×10⁻⁷M), or DBcAMP (1×10⁻⁵M)]for the following 7 days. Even after nicotine treatment was stopped,PTHrP receptor (a), PPARγ (b), and ADRP (c) expression continued to besignificantly lower in the 7d nicotine-only treatment group compared tountreated controls. Treatment with RGZ, PTHrP, or cAMP reversednicotine-induced decrease in PTHrP receptor expression (a: <0.05 vs.control and #<0.001 vs. nicotine; n=3) (FIG. 26A), PPARγ (b: * <0.01 vs.control and #<0.05 vs. nicotine; n=3) (FIG. 26B), and ADRP expression(c: * <0.01 vs. control and #<0.001 vs. nicotine; n=3) (FIG. 26C). Thevalues are mean±SD and n=3. Representative Western blots anddensitometric histograms are shown.

FIGS. 27A and 27B: Embryonic human lung fibroblasts were initiallyexposed to nicotine (10⁻⁹M) for 7 days and then treated with PPARγagonists [rosiglitazone (RGZ) (1×10⁻⁵ M), PTHrP (5×10⁻⁷ M), or DBcAMP(1×10⁻⁵ M)] for the following 7 days. Even after nicotine treatment wasstopped, SMA (a) and calponin (b) expression continued to besignificantly higher in the 7d nicotine-only treatment group compared tountreated controls. Treatment with RGZ, PTHrP, or DBcAMP reversednicotine-induced increase in SMA (a: *p<0.01 vs. control and ^(#)p<0.001vs. nicotine; n=3) (FIG. 27A) and calponin expression (b: *p<0.001 vs.control and ^(#)p<0.001 vs. nicotine; n=3) (FIG. 27B). The values aremean±SD and n=3. Representative Western blots and densitometrichistograms are shown.

FIG. 28 shows representative immunofluorescence staining for ADRP orlipid droplets (red staining) and SMA (green staining) in cultured WI38cells under different experimental conditions. Embryonic human lungfibroblasts were maintained in culture in two-well slides either with orwithout nicotine (10⁻⁹ M) treatment for 7 days and then treated withPPARγ agonists [rosiglitazone (RGZ) (1×10⁻⁵M) or DBcAMP (1×10⁻⁵M)] forthe following 7 days. The 7d nicotine-only treatment group showed amarked decrease in ADRP and lipid droplet staining and a marked increasein SMA staining compared to untreated controls. Both RGZ and DBcAMPtreatments reversed the nicotine-induced decrease in ADRP and lipiddroplet staining and an increase in SMA staining.

FIG. 29: Embryonic human lung fibroblasts were initially exposed tonicotine (10⁻⁹ M) for 7 days and then treated with PPARγ agonists[rosiglitazone (RGZ) (1×10⁻⁵ M), PTHrP (5×10⁻⁷ M), or DBcAMP (1×10⁻⁵ M)]for the following 24 h. At the end of 24 h the expression of PPARγ, thekey lipogenic nuclear transcription factor, continued to besignificantly lower and the expression of SMA and calponin continued tobe significantly higher in the 7d nicotine-only treatment group comparedto untreated controls. Similar to the 7-day data, there was clearevidence of reversal of nicotine-induced lipofibroblast-to-myofibroblasttransdifferentiation even after only 24 h of treatment with PTHrPsignaling agonists. Representative Western blots are shown

FIG. 30 shows that nicotine treatment of WI38 cells for 7 days resultedin a significant decrease in triolein uptake, which was at leastpartially blocked by treatment with rosiglitazone (RGZ) (1 only 10⁻⁵ M),PTHrP (5×10⁻⁷M), or DBcAMP (1×10⁻⁵ M), all stimulants of PTHrP signalingpathway, for 7 days (*p<0.05 vs. control and ^(#)p<0.05 vs. nicotinegroups for all markers). The values are mean±SD and n=3.

FIG. 31 shows that pretreatment with a specific PPARγy antagonist,GW9662 (1×10⁻⁵ M), completely blocked the molecular protection (increasein PPARγ and a decrease in calponin expression) against nicotine (1×10⁻⁹M for 7 days)-induced lipofibroblast-to-myofibroblasttransdifferentiation by all three PTHrP signaling agonists[rosiglitazone (RGZ) (1×10⁻⁵ M), PTHrP (5×10⁷ M), or DBcAMP (1×10⁻⁵ M),(*p<0.001 vs. control; ^(#)p<0.01 vs. nicotine; ̂p<0.001 vs.nicotine+RGZ; ^(@)p<0.001 vs. nicotine+PTHrP; and ^($)p<0.001 vs.nicotine+DBcAMP groups]. The values are mean±SD and n=3.

DETAILED DESCRIPTION

This invention pertains to the discovery of novel methods of treatingthe effects of nicotine exposure (e.g., smoking) on the mammalianrespiratory system. The data presented herein indicate that exposure tonicotine changes the lung interstitium from a predominantly lipogenicphenotype to a myogenic phenotype. The data indicate that downregulationof Peroxisome Proliferator-Activated Receptor (PPAR)γ expression in thelung mesenchyme follows in utero nicotine exposure. As PPARγ expressionis the key to maintaining the lipogenic phenotype of the mesenchyme, webelieve that up-regulating PPARγ nicotine exposure may completely blocknicotine-induced pulmonary lipo-to-myofibroblast transdifferentiation.

The understanding of this mechanism provides a variety of targets forspecific preventive and therapeutic strategies. In this regard, it isnoted that, the decrease in triolein uptake, the functional hallmark ofthe alveolar interstitial lipofibroblast by nicotine was completelyblocked by either tubocurarine or α-bungarotoxin, but not bymecamylamine, suggesting the specific involvement of the α₇ nAChreceptor subtype in nicotine-induced lipo-to-myofibroblasttransdifferentiation.

It is also demonstrated herein that nicotine-induced LIF-to-MYFtransdifferentiation can be completely prevented by concomitanttreatment with PTHrP, DBcAMP, RGZ, and by transiently overexpressingPPAR. Our data suggest nicotine induces alveolar LIF-to-MYFtransdifferentiation through a mechanism involving downregulation oflipogenic PTHrP-mediated, cAMP-dependent PKA signaling pathway, whichcan be prevented using specific molecular targets.

Thus, for example, in certain embodiments, agents that are PTHrPsignaling pathway agonists, e.g., PTHrP and variants or mimeticsthereof, dibutryl cAMP and variants or mimetics thereof, and variousPPARγ antagonists (e.g., rosiglitazone (RGZ)) can partially or fullyblock and in some cases reverse, nincotine-induced pulmonary lipo- tomyo-fibroblast transdifferentiation. PTHrP signaling agonists agoniststhus provide useful agents for treating or preventing nicotine-inducedpulmonary damage.

Thus, for example, smokers or non-smokers exposed to smoke can betreated with PPARγ agonists, e.g., PGD₂, thiazolidinedione, and the like(e.g., orally, parenterally, or preferably via aerosolized route) toreduce, prevent and/or reverse nicotine-induced lung disease. It isbelieved the use of PTHrP signaling agonists agonists will heal thealveoli and increase lung function.

It is also noted that deleterious effect of nicotine on the lungparenchyma may also be mimicked by pulmonary infection as well asexposure to other cyclic hydocarbons and toxic agents in theenvironment. Therefore, it is believed that administration of PTHrPsignaling agonists can be used to reduce, prevent, or reverse lungdamage in these conditions as well.

In addition, it is believed that PTHrP signaling agonists can also beused for prevention of fibrotic injury to pulmonary tissue. Thus, thePTHrP signaling agonists can be used to protect high risk patients, e.g.those who are placed for the first time on mechanical ventilation and/orwho are otherwise subjected to hyperoxia.

I. PTHrP Signaling Agonists.

A wide variety of PTHrP signaling agonists are known. Such agentsinclude but are not limited to PTHrP itself along with variants andmimetics (e.g., parathyroid hormone-related protein (1-36),PTHrP-(7-34)NH₂, PTHrP singularly substituted with a photoreactiveL-p-benzoylphenylalanine (Bpa) at the first N-terminal positions(Bpa¹-PTHrP), see, e.g., Behar et al. (2000) J. Biol. Chem., 275(1):9-17, PTHrP in which residues 5 and/or 23 are switched with thecorresponding residues of PTH (see, e.g., U.S. Pat. No. 6,362,163, whichis incorporated herein by reference), and the like). Certain PTHrPsignaling agonists include, but are not limited to conformationallyconstrained parathyroid hormone (PTH) analogs and derivatives of thoseanalogs containing PTH polypeptide derivatives containing at least oneGlu or Lys substitution at position 6 and/or 10, with, optionallyinstalled, lactam bridges between the side chains of Lys and Glu. Suchderivatives include derivatives of PTH (1-34), PTH(1-33), PTH(1-32),PTH(1-31), PTH(1-30), PTH(1-29), PTH(1-28), PTH(1-27), PTH(1-26),PTH(1-25), PTH(1-24), PTH(1-23), PTH(1-22), PTH (1-21), PTH(1-20),PTH(1-19), PTH(1-18), PTH(1-17), PTH(1-16), PTH(1-15), PTH(1-14),PTH(1-13), PTH(1-12), PTH(1-11), PTH(1-10) and PTH(1-9) polypeptide asdescribed in U.S. Patent Publication No: 20060229240, which isincorporated herein by reference.

In certain embodiments the PTHrP signaling agonists comprise one or morePPARγ agonists. A large number of PPARγ agonists are known to those ofskill in the art many are clinically approved for certain conditions. Incertain embodiments the PPAR gamma agonist is a thiozolidinedione (TZD).In certain embodiments, the PPAR gamma agonist is a glitazone (e.g.,troglitazone, (Resulin), rosiglitazone, pioglitazone, ciglitazone,englitazone, darglitazone, and the like), farglitazar, phenylaceticacid, GW590735, GW677954, Avandia, Avandamet (avandia+metformin), 15deoxy prostaglandin J2 (15PGJ2), 15-deoxy-delta12,14 PGD2, GW-9662,MCC-555, Muraglitzazr (Bristol-Myers/Merck), Galida tesaglitzazr(AstraZeneca), 677954 (GlaxoSmithKline), MBX-102 (Metabolex), T131(Tularik), LY818 (Eli Lilly/Ligand Pharmaceutical), LY929 (EliLilly/Ligand Pharmaceutical), PLX204 (Plexxikon), and the like. Certainpreferred PPAR gamma agonists include, but are not limited to,rosiglitazone or an analogue thereof.

Other suitable PPARγ agonists include, but are not limited toN-(substituted)carbamoylaryl- and heteroaryl substituted aminopropanoicand butanoic acid compounds (see, e.g., U.S. Pat. No. 6,713,514 which isincorporated herein by reference) as well as others (see, e.g., WO91/07107; WO 92/02520; WO 94/01433; WO 89/08651; JP Kokai 69383/92; U.S.Pat. Nos. 4,287,200; 4,340,605; 4,438,141; 4,444,779; 4,461,902;4,572,912; 4,687,777; 4,703,052; 4,725,610; 4,873,255; 4,897,393;4,897,405; 4,918,091; 4,948,900; 5,002,953; 5,061,717; 5,120,754;5,132,317; 5,194,443; 5,223,522; 5,232,925; 5,260,445; 5,814,647,6,200,998, and U.S. Patent Publications 20030109570, and 2005/0014833,which are all incorporated herein by reference).

In various embodiments the PPAR-gamma agonists can be obtainedcommercially. Alternatively, they are synthesized from commerciallyavailable precursors, and/or purified or isolated from naturallyoccurring sources by known biochemical means. (see, e.g., U.S. Pat. No.6,200,998). Synthetic or semi-synthetic versions or derivatives of PPARγagonists are also useful in the inventive method, as arepharmaceutically acceptable salts of PPARγ agonist compounds associatedwith various anions and cations, including, for example, succinate,glutamate, maleate, fumarate, sodium, magnesium, calcium, hydrochloride,chloride, sulfate, carbonate, or bicarbonate.

It will be appreciated that in certain instances two or more differentPPARγ agonists can be used in the methods described herein. The PPARγagonists described above are intended to be illustrative and notlimiting. Utilizing the teachings provided herein, other PPARγ agonistscan be used to inhibit, prevent, or reverse pulmonary damage.

II. Administration of PTHrP Signaling Agonists Prevent, Inhibit, orReverse Pulmonary Damage.

In certain embodiments, this invention provides methods for theinhibition, prevention, and/or reversal of pulmonary damage, e.g. fromsmoking, other forms of nicotine exposure, chronic inflammation, and thelike.

In certain embodiments, one or more of the conditions described hereinare treated increasing the activity of PPARγ (e.g., by upregulatingPPARγ and/or PPARγ receptors, etc.). In certain embodiments, this isaccomplished simply by administration of one or more PPARγ agonists.

A) Formulations

In certain embodiments in order to carry out the methods of theinvention, the PTHrP signaling agonists (e.g., PPARγ agonists) areadministered, e.g. to a smoker, to a person exposed to second handsmoke, etc. The agent can be administered in the “native” form or, ifdesired, in the form of salts, esters, amides, prodrugs, derivatives,and the like, provided the salt, ester, amide, prodrug or derivative issuitable pharmacologically, i.e., effective in the present method.Salts, esters, amides, prodrugs and other derivatives of the activeagents may be prepared using standard procedures known to those skilledin the art of synthetic organic chemistry and described, for example, byMarch (1992) Advanced Organic Chemistry; Reactions, Mechanisms andStructure, 4th Ed. N.Y. Wiley-Interscience.

For example, acid addition salts are prepared from the free base usingconventional methodology that typically involves reaction with asuitable acid. Generally, the base form of the drug is dissolved in apolar organic solvent such as methanol or ethanol and the acid is addedthereto. The resulting salt either precipitates or may be brought out ofsolution by addition of a less polar solvent. Suitable acids forpreparing acid addition salts include both organic acids, e.g., aceticacid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malicacid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. An acid addition salt may be reconvertedto the free base by treatment with a suitable base. Particularlypreferred acid addition salts of the active agents herein are halidesalts, such as may be prepared using hydrochloric or hydrobromic acids.Conversely, preparation of basic salts of the active agents are preparedin a similar manner using a pharmaceutically acceptable base such assodium hydroxide, potassium hydroxide, ammonium hydroxide, calciumhydroxide, trimethylamine, or the like. Particularly preferred basicsalts include alkali metal salts, e.g., the sodium salt, and coppersalts.

Preparation of esters typically involves functionalization of hydroxyland/or carboxyl groups which may be present within the molecularstructure of the drug. The esters are typically acyl-substitutedderivatives of free alcohol groups, i.e., moieties that are derived fromcarboxylic acids of the formula RCOOH where R is alky, and preferably islower alkyl. Esters can be reconverted to the free acids, if desired, byusing conventional hydrogenolysis or hydrolysis procedures.

Amides and prodrugs may also be prepared using techniques known to thoseskilled in the art or described in the pertinent literature. Forexample, amides may be prepared from esters, using suitable aminereactants, or they may be prepared from an anhydride or an acid chlorideby reaction with ammonia or a lower alkyl amine. Prodrugs are typicallyprepared by covalent attachment of a moiety that results in a compoundthat is therapeutically inactive until modified by an individual'smetabolic system.

The active agents identified herein are useful for parenteral, topical,oral, nasal (or otherwise inhaled), rectal, or local administration,such as by aerosol or transdermally, for prophylactic and/or therapeutictreatment of one or more pathological conditions described herein and/orsymptoms thereof. The pharmaceutical compositions can be administered ina variety of unit dosage forms depending upon the method ofadministration. Suitable unit dosage forms, include, but are not limitedto powders, tablets, pills, capsules, lozenges, suppositories, patches,aerosols, inhalers, nasal sprays, injectibles, implantablesustained-release formulations, lipid complexes, etc.

The active agents of this invention are typically combined with apharmaceutically acceptable carrier (excipient) to form apharmacological composition. Pharmaceutically acceptable carriers cancontain one or more physiologically acceptable compound(s) that act, forexample, to stabilize the composition or to increase or decrease theabsorption of the active agent(s). Physiologically acceptable compoundscan include, for example, carbohydrates, such as glucose, sucrose, ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins, protection and uptake enhancerssuch as lipids, compositions that reduce the clearance or hydrolysis ofthe active agents, or excipients or other stabilizers and/or buffers.

Other physiologically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives that areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art wouldappreciate that the choice of pharmaceutically acceptable carrier(s),including a physiologically acceptable compound depends, for example, onthe route of administration of the active agent(s) and on the particularphysio-chemical characteristics of the active agent(s).

The excipients are preferably sterile and generally free of undesirablematter. These compositions may be sterilized by conventional, well-knownsterilization techniques.

In therapeutic applications, the compositions of this invention areadministered to a patient at risk for or suffering from one or moresymptoms of nicotine-induced and/or pollutant induced pulmonarydisease.” Amounts effective for this use will depend upon the severityof the disease and the general state of the patient's health. Single ormultiple administrations of the compositions may be administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of the active agents of the formulations of this invention toeffectively prevent, ameliorate, or reverse one or more symptoms ofpulmonary damage and/or dysfunction.

In certain preferred embodiments, the active agents of this inventionare administered via inhalation (e.g., as an aerosol), parenterally,orally (e.g. via a lozenge, tablet, capsule, etc.) or as an injectablein accordance with standard methods well known to those of skill in theart. In certain embodiments the PPARγ agonists can also be deliveredthrough the skin using conventional transdermal drug delivery systems,i.e., transdermal “patches” wherein the active agent(s) are typicallycontained within a laminated structure that serves as a drug deliverydevice to be affixed to the skin. In such a structure, the drugcomposition is typically contained in a layer, or “reservoir,”underlying an upper backing layer. It will be appreciated that the term“reservoir” in this context refers to a quantity of “activeingredient(s)” that is ultimately available for delivery to the surfaceof the skin. Thus, for example, the “reservoir” may include the activeingredient(s) in an adhesive on a backing layer of the patch, or in anyof a variety of different matrix formulations known to those of skill inthe art. The patch can contain a single reservoir, or it can containmultiple reservoirs.

In one embodiment, the reservoir comprises a polymeric matrix of apharmaceutically acceptable contact adhesive material that serves toaffix the system to the skin during drug delivery. Examples of suitableskin contact adhesive materials include, but are not limited to,polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates,polyurethanes, and the like. Alternatively, the drug-containingreservoir and skin contact adhesive are present as separate and distinctlayers, with the adhesive underlying the reservoir which, in this case,may be either a polymeric matrix as described above, or it may be aliquid or hydrogel reservoir, or may take some other form. The backinglayer in these laminates, which serves as the upper surface of thedevice, preferably functions as a primary structural element of the“patch” and provides the device with much of its flexibility. Thematerial selected for the backing layer is preferably substantiallyimpermeable to the active agent(s) and any other materials that arepresent.

In another embodiment, one or more components of the solution can beprovided as a “concentrate”, e.g., in a storage container (e.g., in apremeasured volume) ready for dilution, or in a soluble capsule readyfor addition to a volume of water.

The pharmaceutically acceptable compositions in accordance with themethod of the present invention can be formulated and manufactured atmore than one concentration of PPARγ agonist, such that modularincrements of PPARγ agonist can be easily administered within thepreferred dose range for the particular mammal. In general, thepreferred effective dose range of PPARγ agonists, in accordance with thepreferred method, is well below toxic levels.

The foregoing formulations and administration methods are intended to beillustrative and not limiting. It will be appreciated that, using theteaching provided herein, other suitable formulations and modes ofadministration can be readily devised.

B) Effective Dosages.

The PTHrP signaling agonists (e.g., PPARγ agonists) will generally beused in an amount effective to achieve the intended purpose (e.g., toprevent, reduce, or reverse nicotine-induced pulmonary damage and thelike). In certain embodiments the agent(s) utilized in the methods ofthis invention are administered at a dose that is effective to partiallyor fully prevent, inhibit, or reverse nicotine-induced lung damage. Incertain instances, such a dosage is comparable to the dosage thatpartially or fully inhibits the transdifferentiation of lipofibroblaststo myofibroblasts in an otherwise normal mammal subject to hyperoxicconditions (e.g., a statistically significant decrease at the 90%, morepreferably at the 95%, and most preferably at the 98% or 99% confidencelevel). The compounds can also be used prophalactically at the same doselevels.

Typically, the PTHrP signaling agonists (e.g., PPARγ agonists) areadministered or applied in a therapeutically effective amount. Atherapeutically effective amount is an amount effective to reduce orprevent one or more symptoms characteristic of nicotine-inducedpulmonary damage. Determination of a therapeutically effective amount iswell within the capabilities of those skilled in the art, especially inlight of the detailed disclosure provided herein. Thus, for example, incertain embodiments, a therapeutically effective amount of a PPAR gammaligand (e.g., rosiglitazone) varies from about 0.1 mg/kg to about 100mg/kg, preferably from about 1 mg/kg to about 25 or 50 mg/kg, mostpreferably from about 3 mg/kg to about 20 mg/kg.

In certain embodiments, an initial dosage of about 1 mg/kg daily,preferably from about 1 mg to about 1000 mg per kilogram daily will beeffective. Daily dose ranges can include about 3 mg/kg to about 100mg/kg is preferred, preferably about 3 mg/kg to about 50 mg/kg, and morepreferably about 3 mg/kg to about 25 or 10 mg/kg. The dosages, however,may be varied depending upon the requirements of the patient, theseverity of the condition being treated, and the compound beingemployed. Determination of the proper dosage for a particular situationis within the skill of the art. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under the circumstance is reached. For convenience, thetotal daily dosage may be divided and administered in portions duringthe day if desired.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One skilled inthe art could readily optimize administration to humans based on animaldata.

Dosage amount and interval may be adjusted individually to provideplasma levels of the inhibitors which are sufficient to maintaintherapeutic effect.

Dosages for typical therapeutics, particularly for PPARγ agonists, areknown to those of skill in the art. Moreover, such dosages are typicallyadvisorial in nature and may be adjusted depending on the particulartherapeutic context, patient tolerance, etc. Single or multipleadministrations of the compositions may be administered depending on thedosage and frequency as required and tolerated by the patient.

In cases of local administration or selective uptake, the effectivelocal concentration of the inhibitors may not be related to plasmaconcentration. One skilled in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.The amount of inhibitor administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

The therapy may be repeated intermittently. The therapy may be providedalone or in combination with other drugs and/or procedures.

C) Toxicity.

Preferably, a therapeutically effective dose of the PTHrP signalingagonists (e.g., PPARγ agonists) described herein will providetherapeutic benefit without causing substantial toxicity.

Toxicity of the PPARγ agonists described herein can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., by determining the LD₅₀ (the dose lethal to 50% of thepopulation) or the LD₁₀₀ (the dose lethal to 100% of the population). Itis noted that toxicity of numerous PPARγ agonists ligands is wellcharacterized. The dose ratio between toxic and therapeutic effect isthe therapeutic index. Inhibitors which exhibit high therapeutic indicesare preferred. The data obtained from these cell culture assays andanimal studies can be used in formulating a dosage range that is nottoxic for use in human. The dosage of the inhibitors described hereinlies preferably within a range of circulating concentrations thatinclude the effective dose with little or no toxicity. The dosage mayvary within this range depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See, e.g., Fingl et al. (1975) In: ThePharmacological Basis of Therapeutics, Ch. 1, p. 1).

III. Screening for Agents that Inhibit, Prevent, or ReverseNicotine-Induced Pulmonary Damage.

As indicated above, in one aspect, this invention pertains to thediscovery of a mechanism of nicotine-induced pulmonary damage and thatPTHrP signaling agonists (e.g., PPARγ agonists) can inhibit, prevent,and/or reverse such damage. Thus methods of screening for PPARγ agonistsprovide good methods for screening for agents that can inhibit, prevent,and/or reverse nicotine-induced pulmonary damage.

When screening for PPARγ agonists, a positive assay result need notindicate that a particular test agent is a good pharmaceutical. Rather apositive test result can simply an indicator that the tested compound isa good potential agent and/or can serve as a lead compound in thedevelopment of other clinically relevant agonists.

Using known activities, and/or nucleic acid sequences, and/or amino acidsequences of PTHrP, PPARγ and/or the PPARγ receptor, expression level(s)and/or activity of a test compound can readily be determined accordingto a number of different methods, e.g., as described below. Inparticular, expression levels of PPARγ and/or PPARγ receptors can bealtered by changes in the copy number of the gene(s) encoding thosecomponents, and/or by changes in the transcription of the gene product(i.e. transcription of mRNA), and/or by changes in translation of thegene product (i.e. translation of the protein), and/or bypost-translational modification(s) (e.g. protein folding, glycosylation,etc.). Thus useful assays of this invention include assaying for copynumber, level of transcribed mRNA, level of translated protein, activityof translated protein, etc.

A) Nucleic-Acid Based Assays.

1) Target Molecules.

Changes in expression level(s) of PTHrP signaling pathway components,e.g., PPARγ and/or the PPARγ receptor can be detected by measuringchanges in mRNA encoding such component(s) and/or a nucleic acid derivedfrom the mRNA (e.g. reverse-transcribed cDNA, etc.). In order to measurethe expression level it is desirable to provide a nucleic acid samplefor such analysis. In preferred embodiments the nucleic acid is found inor derived from a biological sample. The term “biological sample”, asused herein, refers to a sample obtained from an organism or fromcomponents (e.g., cells) of an organism, or from cells in culture. Thesample may be of any biological tissue or fluid. Biological samples mayalso include organs or sections of tissues such as frozen sections takenfor histological purposes.

The nucleic acid (e.g., mRNA nucleic acid derived from mRNA) is, incertain preferred embodiments, isolated from the sample according to anyof a number of methods well known to those of skill in the art. Methodsof isolating mRNA are well known to those of skill in the art. Forexample, methods of isolation and purification of nucleic acids aredescribed in detail in by Tijssen ed., (1993) Chapter 3 of LaboratoryTechniques in Biochemistry and Molecular Biology: Hybridization WithNucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation,Elsevier, N.Y. and Tijssen ed.

In a preferred embodiment, the “total” nucleic acid is isolated from agiven sample using, for example, an acid guanidinium-phenol-chloroformextraction method and polyA+mRNA is isolated by oligo dT columnchromatography or by using (dT)n magnetic beads (see, e.g., Sambrook etal., (1989) Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3,Cold Spring Harbor Laboratory, or Current Protocols in MolecularBiology, F. Ausubel et al., ed. Greene Publishing andWiley-Interscience, New York (1987)).

Frequently, it is desirable to amplify the nucleic acid sample prior toassaying for expression level. Methods of amplifying nucleic acids arewell known to those of skill in the art and include, but are not limitedto polymerase chain reaction (PCR, see. e.g., Innis, et al., (1990) PCRProtocols. A guide to Methods and Application. Academic Press, Inc. SanDiego,), ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics4: 560, Landegren et al. (1988) Science 241: 1077, and Barringer et al.(1990) Gene 89: 117, transcription amplification (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequencereplication (Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874),dot PCR, and linker adapter PCR, etc.).

In certain embodiments, where it is desired to quantify thetranscription level (and thereby expression) of PPAR gamma and/or thePPAR gamma receptor in a sample, the nucleic acid sample is one in whichthe concentration of the mRNA transcript(s), or the concentration of thenucleic acids derived from the mRNA transcript(s), is proportional tothe transcription level (and therefore expression level) of the gene(s)of interest. Similarly, it is preferred that the hybridization signalintensity be proportional to the amount of hybridized nucleic acid.While it is preferred that the proportionality be relatively strict(e.g., a doubling in transcription rate results in a doubling in mRNAtranscript in the sample nucleic acid pool and a doubling inhybridization signal), one of skill will appreciate that theproportionality can be more relaxed and even non-linear. Thus, forexample, an assay where a 5 fold difference in concentration of thetarget mRNA results in a 3 to 6 fold difference in hybridizationintensity is sufficient for most purposes.

Where more precise quantification is required appropriate controls canbe run to correct for variations introduced in sample preparation andhybridization as described herein. In addition, serial dilutions of“standard” target nucleic acids (e.g., mRNAs) can be used to preparecalibration curves according to methods well known to those of skill inthe art. Of course, where simple detection of the presence or absence ofa transcript or large differences of changes in nucleic acidconcentration is desired, no elaborate control or calibration isrequired.

In the simplest embodiment, the nucleic acid sample is the total mRNA ora total cDNA isolated and/or otherwise derived from a biological sample(e.g. a neurological cell or tissue). The nucleic acid may be isolatedfrom the sample according to any of a number of methods well known tothose of skill in the art as indicated above.

2) Hybridization-Based Assays.

Using the known sequences for PPAR gamma and/or the PPAR gamma receptor,detecting and/or quantifying the transcript(s) can be routinelyaccomplished using nucleic acid hybridization techniques (see, e.g.,Sambrook et al. supra). For example, one method for evaluating thepresence, absence, or quantity of reverse-transcribed cDNA involves a“Southern Blot”. In a Southern Blot, the DNA (e.g., reverse-transcribedmRNA), typically fragmented and separated on an electrophoretic gel, ishybridized to a probe specific for subject nucleic acid(s) (or to amutant thereof). Comparison of the intensity of the hybridization signalfrom the probe with a “control” probe (e.g. a probe for a “housekeepinggene) provides an estimate of the relative expression level of thetarget nucleic acid.

Alternatively, the mRNA of interest can be directly quantified in aNorthern blot. In brief, the mRNA is isolated from a given cell sampleusing, for example, an acid guanidinium-phenol-chloroform extractionmethod. The mRNA is then electrophoresed to separate the mRNA speciesand the mRNA is transferred from the gel to a nitrocellulose membrane.As with the Southern blots, labeled probes are used to identify and/orquantify the target PPAR gamma and/or the PPAR gamma receptor mRNA.Appropriate controls (e.g., probes to housekeeping genes) provide areference for evaluating relative expression level.

An alternative means for determining the expression level(s) of PPARgamma and/or the PPAR gamma receptor is in situ hybridization. In situhybridization assays are well known (e.g., Angerer (1987) Meth. Enzymol152: 649). Generally, in situ hybridization comprises the followingmajor steps: (1) fixation of tissue or biological structure to beanalyzed; (2) prehybridization treatment of the biological structure toincrease accessibility of target DNA, and to reduce nonspecific binding;(3) hybridization of the mixture of nucleic acids to the nucleic acid inthe biological structure or tissue; (4) post-hybridization washes toremove nucleic acid fragments not bound in the hybridization and (5)detection of the hybridized nucleic acid fragments. The reagent used ineach of these steps and the conditions for use vary depending on theparticular application.

In some applications it is necessary to block the hybridization capacityof repetitive sequences. Thus, in some embodiments, tRNA, human genomicDNA, or Cot-1 DNA is used to block non-specific hybridization.

3) Amplification-Based Assays.

In another embodiment, amplification-based assays can be used to measureexpression (transcription) level of PTHrP signaling pathway componentse.g., PPAR gamma and/or the PPAR gamma receptor. In suchamplification-based assays, the target nucleic acid sequences (PPARgamma and/or the PPAR gamma receptor nucleic acids) act as template(s)in amplification reaction(s) (e.g. Polymerase Chain Reaction (PCR) orreverse-transcription PCR (RT-PCR)). In a quantitative amplification,the amount of amplification product will be proportional to the amountof template in the original sample. Comparison to appropriate (e.g.tissue or cells unexposed to the test agent) controls provides a measureof the target transcript level.

Methods of “quantitative” amplification are well known to those of skillin the art. For example, quantitative PCR involves simultaneouslyco-amplifying a known quantity of a control sequence using the sameprimers. This provides an internal standard that may be used tocalibrate the PCR reaction. Detailed protocols for quantitative PCR areprovided in Innis et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc. N.Y.). One approach, for example,involves simultaneously co-amplifying a known quantity of a controlsequence using the same primers as those used to amplify the target.This provides an internal standard that may be used to calibrate the PCRreaction.

One typical internal standard is a synthetic AW106 cRNA. The AW106 cRNAis combined with RNA isolated from the sample according to standardtechniques known to those of skill in the art. The RNA is then reversetranscribed using a reverse transcriptase to provide copy DNA. The cDNAsequences are then amplified (e.g., by PCR) using labeled primers. Theamplification products are separated, typically by electrophoresis, andthe amount of labeled nucleic acid (proportional to the amount ofamplified product) is determined. The amount of mRNA in the sample isthen calculated by comparison with the signal produced by the knownAW106 RNA standard. Detailed protocols for quantitative PCR are providedin PCR Protocols, A Guide to Methods and Applications, Innis et al.(1990) Academic Press, Inc. N.Y. The known nucleic acid sequence(s) forPPAR gamma and/or the PPAR gamma receptor are sufficient to enable oneof skill to routinely select primers to amplify any portion of thegene(s).

4) Hybridization Formats and Optimization of Hybridization Conditions.

i) Array-Based Hybridization Formats.

In one embodiment, the methods of this invention can be utilized inarray-based hybridization formats. Arrays are a multiplicity ofdifferent “probe” or “target” nucleic acids (or other compounds)attached to one or more surfaces (e.g., solid, membrane, or gel). In apreferred embodiment, the multiplicity of nucleic acids (or othermoieties) is attached to a single contiguous surface or to amultiplicity of surfaces juxtaposed to each other.

In an array format a large number of different hybridization reactionscan be run essentially “in parallel.” This provides rapid, essentiallysimultaneous, evaluation of a number of hybridizations in a single“experiment”. Methods of performing hybridization reactions in arraybased formats are well known to those of skill in the art (see, e.g.,Pastinen (1997) Genome Res. 7: 606-614; Jackson (1996) NatureBiotechnology 14:1685; Chee (1995) Science 274: 610; WO 96/17958, Pinkelet al. (1998) Nature Genetics 20: 207-211).

Arrays, particularly nucleic acid arrays can be produced according to awide variety of methods well known to those of skill in the art. Forexample, in a simple embodiment, “low density” arrays can simply beproduced by spotting (e.g. by hand using a pipette) different nucleicacids at different locations on a solid support (e.g. a glass surface, amembrane, etc.).

This simple spotting, approach has been automated to produce highdensity spotted arrays (see, e.g., U.S. Pat. No. 5,807,522). This patentdescribes the use of an automated system that taps a microcapillaryagainst a surface to deposit a small volume of a biological sample. Theprocess is repeated to generate high-density arrays.

Arrays can also be produced using oligonucleotide synthesis technology.Thus, for example, U.S. Pat. No. 5,143,854 and PCT Patent PublicationNos. WO 90/15070 and 92/10092 teach the use of light-directedcombinatorial synthesis of high density oligonucleotide arrays.Synthesis of high-density arrays is also described in U.S. Pat. Nos.5,744,305, 5,800,992 and 5,445,934.

ii) Other Hybridization Formats.

As indicated above a variety of nucleic acid hybridization formats areknown to those skilled in the art. For example, common formats includesandwich assays and competition or displacement assays. Such assayformats are generally described in Hames and Higgins (1985) Nucleic AcidHybridization, A Practical Approach, IRL Press; Gall and Pardue (1969)Proc. Natl. Acad. Sci. USA 63: 378-383; and John et al. (1969) Nature223: 582-587.

Sandwich assays are commercially useful hybridization assays fordetecting or isolating nucleic acid sequences. Such assays utilize a“capture” nucleic acid covalently immobilized to a solid support and alabeled “signal” nucleic acid in solution. The sample will provide thetarget nucleic acid. The “capture” nucleic acid and “signal” nucleicacid probe hybridize with the target nucleic acid to form a “sandwich”hybridization complex. To be most effective, the signal nucleic acidshould not hybridize with the capture nucleic acid.

Typically, labeled signal nucleic acids are used to detecthybridization. Complementary nucleic acids or signal nucleic acids maybe labeled by any one of several methods typically used to detect thepresence of hybridized polynucleotides. The most common method ofdetection is the use of autoradiography with ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P-labelled probes or the like. Other labels include ligands that bindto labeled antibodies, fluorophores, chemi-luminescent agents, enzymes,and antibodies that can serve as specific binding pair members for alabeled ligand.

Detection of a hybridization complex may require the binding of a signalgenerating complex to a duplex of target and probe polynucleotides ornucleic acids. Typically, such binding occurs through ligand andanti-ligand interactions as between a ligand-conjugated probe and ananti-ligand conjugated with a signal.

The sensitivity of the hybridization assays may be enhanced through useof a nucleic acid amplification system that multiplies the targetnucleic acid being detected. Examples of such systems include thepolymerase chain reaction (PCR) system and the ligase chain reaction(LCR) system. Other methods recently described in the art are thenucleic acid sequence based amplification (NASBAO, Cangene, Mississauga,Ontario) and Q Beta Replicase systems.

iii) Optimization of Hybridization Conditions.

Nucleic acid hybridization simply involves providing a denatured probeand target nucleic acid under conditions where the probe and itscomplementary target can form stable hybrid duplexes throughcomplementary base pairing. The nucleic acids that do not form hybridduplexes are then washed away leaving the hybridized nucleic acids to bedetected, typically through detection of an attached detectable label.It is generally recognized that nucleic acids are denatured byincreasing the temperature or decreasing the salt concentration of thebuffer containing the nucleic acids, or in the addition of chemicalagents, or the raising of the pH. Under low stringency conditions (e.g.,low temperature and/or high salt and/or high target concentration)hybrid duplexes (e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form evenwhere the annealed sequences are not perfectly complementary. Thusspecificity of hybridization is reduced at lower stringency. Conversely,at higher stringency (e.g., higher temperature or lower salt) successfulhybridization requires fewer mismatches.

One of skill in the art will appreciate that hybridization conditionsmay be selected to provide any degree of stringency. In a preferredembodiment, hybridization is performed at low stringency to ensurehybridization and then subsequent washes are performed at higherstringency to eliminate mismatched hybrid duplexes. Successive washesmay be performed at increasingly higher stringency (e.g., down to as lowas 0.25×SSPE at 37° C. to 70° C.) until a desired level of hybridizationspecificity is obtained. Stringency can also be increased by addition ofagents such as formamide. Hybridization specificity may be evaluated bycomparison of hybridization to the test probes with hybridization to thevarious controls that can be present.

In general, there is a tradeoff between hybridization specificity(stringency) and signal intensity. Thus, in a preferred embodiment, thewash is performed at the highest stringency that produces consistentresults and that provides a signal intensity greater than approximately10% of the background intensity. Thus, in a preferred embodiment, thehybridized array may be washed at successively higher stringencysolutions and read between each wash. Analysis of the data sets thusproduced will reveal a wash stringency above which the hybridizationpattern is not appreciably altered and which provides adequate signalfor the particular probes of interest.

In a preferred embodiment, background signal is reduced by the use of ablocking reagent (e.g., tRNA, sperm DNA, cot-1 DNA, etc.) during thehybridization to reduce non-specific binding. The use of blocking agentsin hybridization is well known to those of skill in the art (see, e.g.,Chapter 8 in P. Tijssen, supra.).

Methods of optimizing hybridization conditions are well known to thoseof skill in the art (see, e.g., Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 24: Hybridization With NucleicAcid Probes, Elsevier, N.Y.).

Optimal conditions are also a function of the sensitivity of label(e.g., fluorescence) detection for different combinations of substratetype, fluorochrome, excitation and emission bands, spot size and thelike. Low fluorescence background surfaces can be used (see, e.g., Chu(1992) Electrophoresis 13: 105-114). The sensitivity for detection ofspots (“target elements”) of various diameters on the candidate surfacescan be readily determined by, e.g., spotting a dilution series offluorescently end labeled DNA fragments. These spots are then imagedusing conventional fluorescence microscopy. The sensitivity, linearity,and dynamic range achievable from the various combinations offluorochrome and solid surfaces (e.g., glass, fused silica, etc.) canthus be determined. Serial dilutions of pairs of fluorochrome in knownrelative proportions can also be analyzed. This determines the accuracywith which fluorescence ratio measurements reflect actual fluorochromeratios over the dynamic range permitted by the detectors andfluorescence of the substrate upon which the probe has been fixed.

iv) Labeling and Detection of Nucleic Acids.

The probes used herein for detection of PPARγ and/or the PPARγ receptorexpression levels can be full length or less than the full length of thetarget nucleic acid. Shorter probes are empirically tested forspecificity. Preferred probes are sufficiently long so as tospecifically hybridize with the target nucleic acid(s) under stringentconditions. The preferred size range is from about 10, 15, or 20 basesto the length of the target mRNA, more preferably from about 30 bases tothe length of the target mRNA, and most preferably from about 40 basesto the length of the target mRNA. The probes are typically labeled, witha detectable label as described above.

B) Detection of Expressed Protein

1) Assay Formats.

In addition to, or in alternative to, the detection of PTHrP signalingpathway components, e.g., PPAR gamma and/or the PPAR gamma receptornucleic acid(s), alterations in expression of PPAR gamma and/or the PPARgamma receptor can be detected and/or quantified by detecting and/orquantifying the amount and/or activity of translated PPAR gamma proteinand/or the PPAR gamma receptor protein(s).

The expression of PPAR gamma and/or the PPAR gamma receptor can bedetected and quantified by any of a number of methods well known tothose of skill in the art. These can include analytic biochemicalmethods such as electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, and the like, or variousimmunological methods such as fluid or gel precipitin reactions,immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, western blotting, and the like.

In one embodiment, the PPAR gamma and/or the PPAR gamma receptor aredetected/quantified in an electrophoretic protein separation (e.g., a 1-or 2-dimensional electrophoresis). Means of detecting proteins usingelectrophoretic techniques are well known to those of skill in the art(see generally, R. Scopes (1982) Protein Purification, Springer-Verlag,N.Y.; Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to ProteinPurification, Academic Press, Inc., N.Y.).

In another embodiment, Western blot (immunoblot) analysis is used todetect and quantify the presence of PPAR gamma and/or the PPAR gammareceptor in the sample. This technique generally comprises separatingsample proteins by gel electrophoresis on the basis of molecular weight,transferring the separated proteins to a suitable solid support, (suchas a nitrocellulose filter, a nylon filter, or derivatized nylonfilter), and incubating the sample with the antibodies that specificallybind the target polypeptide(s).

The antibodies specifically bind to the target member, e.g.,polypeptide(s), and can be directly labeled or alternatively may besubsequently detected using labeled antibodies (e.g., labeled sheepanti-mouse antibodies) that specifically bind to a domain of theantibody.

In certain embodiments, the PPAR gamma and/or the PPAR gamma receptorare detected using an immunoassay. As used herein, an immunoassay is anassay that utilizes an antibody to specifically bind to the analyte(e.g., the target polypeptide(s), such as PPAR gamma and/or the PPARgamma receptor). The immunoassay is thus characterized by detection ofspecific binding of a polypeptide of this invention to an antibody asopposed to the use of other physical or chemical properties to isolate,target, and quantify the analyte.

Any of a number of well recognized immunological binding assays (see,e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168) arewell suited to detection or quantification of the polypeptide(s)identified herein. For a review of the general immunoassays, see alsoAsai (1993) Methods in Cell Biology Volume 37: Antibodies in CellBiology, Academic Press, Inc. New York; Stites & Ten (1991) Basic andClinical Immunology 7th Edition.

Immunological binding assays (or immunoassays) typically utilize a“capture agent” to specifically bind to and often immobilize the analyte(e.g., PPAR gamma and/or the PPAR gamma receptor). In certainembodiments, the capture agent is an antibody.

Immunoassays also often utilize a labeling agent to specifically bind toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibody/analyte complex. Thus, the labeling agent may be a labeledpolypeptide or a labeled antibody that specifically recognizes thealready bound target polypeptide. Alternatively, the labeling agent maybe a third moiety, such as another antibody, that specifically binds tothe capture agent/polypeptide complex.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, andAkerstrom (1985) J. Immunol., 135: 2589-2542).

Typical immunoassays for detecting the target polypeptide(s), e.g., PPARgamma and/or the PPAR gamma receptor, are either competitive ornoncompetitive. Noncompetitive immunoassays are assays in which theamount of captured analyte is directly measured. In one “sandwich”assay, for example, the capture agents (antibodies) can be bounddirectly to a solid substrate where they are immobilized. Theseimmobilized antibodies then capture the target polypeptide present inthe test sample. The target polypeptide thus immobilized is then boundby a labeling agent, such as a second antibody bearing a label.

In competitive assays, the amount of analyte (e.g., PPAR gamma and/orthe PPAR gamma receptor) present in the sample is measured indirectly bymeasuring the amount of an added (exogenous) analyte displaced (orcompeted away) from a capture agent (antibody) by the analyte present inthe sample. In one competitive assay, a known amount of, in this case,labeled polypeptide is added to the sample and the sample is thencontacted with a capture agent. The amount of labeled polypeptide boundto the antibody is inversely proportional to the concentration of targetpolypeptide present in the sample.

In one embodiment, the antibody is immobilized on a solid substrate. Theamount of target polypeptide bound to the antibody may be determinedeither by measuring the amount of target polypeptide present in apolypeptide/antibody complex, or alternatively by measuring the amountof remaining uncomplexed polypeptide.

The immunoassay methods of the present invention include an enzymeimmunoassay (EIA) which utilizes, depending on the particular protocolemployed, unlabeled or labeled (e.g., enzyme-labeled) derivatives ofpolyclonal or monoclonal antibodies or antibody fragments orsingle-chain antibodies that bind PPAR gamma and/or the PPAR gammareceptor, either alone or in combination. In the case where the antibodythat binds a PPAR gamma and/or the PPAR gamma receptor is not labeled, adifferent detectable marker, for example, an enzyme-labeled antibodycapable of binding to the monoclonal antibody which binds the PPAR gammaand/or the PPAR gamma receptor, may be employed. Any of the knownmodifications of EIA, for example, enzyme-linked immunoabsorbent assay(ELISA), may also be employed. As indicated above, also contemplated bythe present invention are immunoblotting immunoassay techniques such aswestern blotting employing an enzymatic detection system.

The immunoassay methods of the present invention may also be other knownimmunoassay methods, for example, fluorescent immunoassays usingantibody conjugates or antigen conjugates of fluorescent substances suchas fluorescein or rhodamine, latex agglutination with antibody-coated orantigen-coated latex particles, haemagglutination with antibody-coatedor antigen-coated red blood corpuscles, and immunoassays employing anavidin-biotin or strepavidin-biotin detection systems, and the like.

The particular parameters employed in the immunoassays of the presentinvention can vary widely depending on various factors such as theconcentration of antigen in the sample, the nature of the sample, thetype of immunoassay employed and the like. Optimal conditions can bereadily established by those of ordinary skill in the art. In certainembodiments, the amount of antibody that binds PPAR gamma and/or thePPAR gamma receptor is typically selected to give 50% binding ofdetectable marker in the absence of sample. If purified antibody is usedas the antibody source, the amount of antibody used per assay willgenerally range from about 1 ng to about 100 ng. Typical assayconditions include a temperature range of about 4° C. to about 45° C.,preferably about 25° C. to about 37° C., and most preferably about 25°C., a pH value range of about 5 to 9, preferably about 7, and an ionicstrength varying from that of distilled water to that of about 0.2Msodium chloride, preferably about that of 0.15M sodium chloride. Timeswill vary widely depending upon the nature of the assay, and generallyrange from about 0.1 minute to about 24 hours. A wide variety ofbuffers, for example PBS, may be employed, and other reagents such assalt to enhance ionic strength, proteins such as serum albumins,stabilizers, biocides and non-ionic detergents may also be included.

The assays of this invention are scored (as positive or negative orquantity of target polypeptide) according to standard methods well knownto those of skill in the art. The particular method of scoring willdepend on the assay format and choice of label. For example, a WesternBlot assay can be scored by visualizing the colored product produced bythe enzymatic label. A clearly visible colored band or spot at thecorrect molecular weight is scored as a positive result, while theabsence of a clearly visible spot or band is scored as a negative. Theintensity of the band or spot can provide a quantitative measure oftarget polypeptide concentration.

Antibodies for use in the various immunoassays described herein can beroutinely produced as described below.

2) Antibodies to PTHrP Signaling Pathway Components (e.g., PPAR Gammaand/or the PPAR Gamma Receptor.

Either polyclonal or monoclonal antibodies can be used in theimmunoassays of the invention described herein. Polyclonal antibodiesare typically raised by multiple injections (e.g. subcutaneous orintramuscular injections) of substantially pure polypeptides orantigenic polypeptides into a suitable non-human mammal. Theantigenicity of the target peptides can be determined by conventionaltechniques to determine the magnitude of the antibody response of ananimal that has been immunized with the peptide. Generally, the peptidesthat are used to raise antibodies for use in the methods of thisinvention should generally be those which induce production of hightiters of antibody with relatively high affinity for targetpolypeptides, such as PPAR gamma and/or the PPAR gamma receptor.

If desired, the immunizing peptide can be coupled to a carrier proteinby conjugation using techniques that are well-known in the art. Suchcommonly used carriers which are chemically coupled to the peptideinclude keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serumalbumin (BSA), and tetanus toxoid. The coupled peptide is then used toimmunize the animal (e.g. a mouse or a rabbit).

The antibodies are then obtained from blood samples taken from themammal. The techniques used to develop polyclonal antibodies are knownin the art (see, e.g., Methods of Enzymology, “Production of AntiseraWith Small Doses of Immunogen: Multiple Intradermal Injections”,Langone, et al. eds. (Acad. Press, 1981)). Polyclonal antibodiesproduced by the animals can be further purified, for example, by bindingto and elution from a matrix to which the peptide to which theantibodies were raised is bound. Those of skill in the art will know ofvarious techniques common in the immunology arts for purification and/orconcentration of polyclonal antibodies, as well as monoclonal antibodiessee, for example, Coligan, et al. (1991) Unit 9, Current Protocols inImmunology, Wiley Interscience).

In certain embodiments, however, the antibodies produced will bemonoclonal antibodies (“mAb's”). For preparation of monoclonalantibodies, immunization of a mouse or rat is preferred. The term“antibody” as used in this invention includes intact molecules as wellas fragments thereof, such as, Fab and F(ab′)^(2′), and/or single-chainantibodies (e.g. scFv) which are capable of binding an epitopicdeterminant.

The general method used for production of hybridomas secreting mAbs iswell known (Kohler and Milstein (1975) Nature, 256:495). Briefly, asdescribed by Kohler and Milstein the technique comprised isolatinglymphocytes from regional draining lymph nodes of five separate cancerpatients with either melanoma, teratocarcinoma or cancer of the cervix,glioma or lung, (where samples were obtained from surgical specimens),pooling the cells, and fusing the cells with SHFP-1. Hybridomas werescreened for production of antibody which bound to cancer cell lines.Confirmation of specificity among mAb's can be accomplished usingrelatively routine screening techniques (such as the enzyme-linkedimmunosorbent assay, or “ELISA”) to determine the elementary reactionpattern of the mAb of interest.

Antibody fragments, e.g. single chain antibodies (scFv or others), canalso be produced/selected using phage display technology. The ability toexpress antibody fragments on the surface of viruses that infectbacteria (bacteriophage or phage) makes it possible to isolate a singlebinding antibody fragment, e.g., from a library of greater than 10¹⁰nonbinding clones. To express antibody fragments on the surface of phage(phage display), an antibody fragment gene is inserted into the geneencoding a phage surface protein (e.g., pIII) and the antibodyfragment-pIII fusion protein is displayed on the phage surface(McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboom et al. (1991)Nucleic Acids Res. 19: 4133-4137).

Since the antibody fragments on the surface of the phage are functional,phage bearing antigen binding antibody fragments can be separated fromnon-binding phage by antigen affinity chromatography (McCafferty et al.(1990) Nature, 348: 552-554). Depending on the affinity of the antibodyfragment, enrichment factors of 20 fold-1,000,000 fold are obtained fora single round of affinity selection. By infecting bacteria with theeluted phage, however, more phage can be grown and subjected to anotherround of selection. In this way, an enrichment of 1000 fold in one roundcan become 1,000,000 fold in two rounds of selection (McCafferty et al.(1990) Nature, 348: 552-554). Thus even when enrichments are low (Markset al. (1991) J. Mol. Biol. 222: 581-597), multiple rounds of affinityselection can lead to the isolation of rare phage. Since selection ofthe phage antibody library on antigen results in enrichment, themajority of clones bind antigen after as few as three to four rounds ofselection. Thus only a relatively small number of clones (severalhundred) need to be analyzed for binding to antigen.

Human antibodies can be produced without prior immunization bydisplaying very large and diverse V-gene repertoires on phage (Marks etal. (1991) J. Mol. Biol. 222: 581-597). In one embodiment natural V_(H)and V_(L) repertoires present in human peripheral blood lymphocytes arewere isolated from unimmunized donors by PCR. The V-gene repertoireswere spliced together at random using PCR to create a scFv generepertoire which is was cloned into a phage vector to create a libraryof 30 million phage antibodies (Id.). From this single “naive” phageantibody library, binding antibody fragments have been isolated againstmore than 17 different antigens, including haptens, polysaccharides andproteins (Marks et al. (1991) J. Mol. Biol. 222: 581-597; Marks et al.(1993). Bio/Technology. 10: 779-783; Griffiths et al. (1993) EMBO J. 12:725-734; Clackson et al. (1991) Nature. 352: 624-628). Antibodies havebeen produced against self proteins, including human thyroglobulin,immunoglobulin, tumor necrosis factor and CEA (Griffiths et al. (1993)EMBO J. 12: 725-734). It is also possible to isolate antibodies againstcell surface antigens by selecting directly on intact cells. Theantibody fragments are highly specific for the antigen used forselection and have affinities in the 1:M to 100 nM range (Marks et al.(1991) J. Mol. Biol. 222: 581-597; Griffiths et al. (1993) EMBO J. 12:725-734). Larger phage antibody libraries result in the isolation ofmore antibodies of higher binding affinity to a greater proportion ofantigens.

It will also be recognized that antibodies can be prepared by any of anumber of commercial services (e.g., Berkeley antibody laboratories,Bethyl Laboratories, Anawa, Eurogenetec, etc.).

C) Assays for Activity

Another aspect of the invention is a method of assaying a compound thatincreases PTHrP signaling, e.g., activates/agonizes PPARγ, by forexample, increasing PPARγ activity (e.g., by inhibition of Egr-1expression, etc.). Methods of detecting PPARγ activity are known tothose of skill in the art. For example, screening methods utilizinganimal cells having introduced therein reporter plasmid(s) containing areporter gene linked to a PPAR expression vector and a PPAR responseelement (PPRE), are described in PCT Publication WO 96/22884 and byTontonoz et al. (1994) Genes and Development, 8: 1224-1234, which areincorporated herein by reference. Another approach utilizes animal cellshaving introduced therein a vector for expressing fused protein in whichthe DNA binding domain of GAL4 (a yeast transcription factor) and theligand binding domain of PPAR linked together, along with an introducedreporter plasmid containing a reporter gene linked to the responseelement of GAL4 (GAL4 binding element), see, e.g., PCT Publication WO96/33724; Lehmann et al. (1995) J. Biol. Chem., 270: 12953-12956;Willson et al. (1996) J. Med. Chem., 39: 665-668 which are incorporatedherein by reference. Another method for directly detecting the bindingbetween PPAR and a ligand without using any animal cell or reporter geneexamined binding and antagonism between a fused protein comprising theligand binding domain of PPAR and glutathione-S-transferase (GST) and atest compound labeled with a radioisotope (see, e.g., Willson et al.(1996) J. Med. Chem., 39: 665-668; Buckle et al. (1996) Bioorganic &Medical Chemistry Letters 6: 2121-2126 which are incorporated herein byreference). Other suitable assays are described by Krey et al., (1997)Mol. Endocrinol., 11: 779-791 and are shown in U.S. Patent Publication2002/0119499, which are incorporated herein by reference

These PPARγ activity assays described herein are intended to beillustrative and not limiting. Using the teachings provided herein,PPARγ activity assays to identify compounds useful to reduce, prevent,or reverse nicotine-induced pulmonary damage will be known to those ofskill in the art.

D) Assay Optimization.

The assays described herein can be optimized for use in particularcontexts, depending, for example, on the source and/or nature of thebiological sample and/or the particular test agents, and/or the analyticfacilities available. Thus, for example, optimization can involvedetermining optimal conditions for binding assays, optimum sampleprocessing conditions (e.g. preferred isolation conditions), antibodyconditions that maximize signal to noise, protocols that improvethroughput, etc. In addition, assay formats can be selected and/oroptimized according to the availability of equipment and/or reagents.Thus, for example, where commercial antibodies or ELISA kits areavailable it may be desired to assay protein concentration.

Routine selection and optimization of assay formats is well known tothose of ordinary skill in the art.

E) Scoring the Assay(s).

The assays of this invention are scored according to standard methodswell known to those of skill in the art. The assays of this inventionare typically scored as positive where there is a difference between theactivity seen with the test agent present or where the test agent hasbeen previously applied, and the (usually negative) control. In certainembodiments, the change is a statistically significant change, e.g. asdetermined using any statistical test suited for the data set provided(e.g. t-test, analysis of variance (ANOVA), semiparametric techniques,non-parametric techniques (e.g. Wilcoxon Mann-Whitney Test, WilcoxonSigned Ranks Test, Sign Test, Kruskal-Wallis Test, etc.). Preferably thestatistically significant change is significant at least at the 85%,more preferably at least at the 90%, still more preferably at least atthe 95%, and most preferably at least at the 98% or 99% confidencelevel). In certain embodiments, the change is at least a 10% change,preferably at least a 20% change, more preferably at least a 50% changeand most preferably at least a 90% change.

IV. Agents for Screening: Combinatorial Libraries (e.g., Small OrganicMolecules)

Virtually any agent can be screened according to the methods of thisinvention. Such agents include, but are not limited to nucleic acids,proteins, sugars, polysaccharides, glycoproteins, lipids, and smallorganic molecules. The term small organic molecules typically refers tomolecules of a size comparable to those organic molecules generally usedin pharmaceuticals. The term excludes biological macromolecules (e.g.,proteins, nucleic acids, etc.). Preferred small organic molecules rangein size up to about 5000 Da, more preferably up to 2000 Da, and mostpreferably up to about 1000 Da.

Conventionally, new chemical entities with useful properties aregenerated by identifying a chemical compound (called a “lead compound”)with some desirable property or activity, creating variants of the leadcompound, and evaluating the property and activity of those variantcompounds. However, the current trend is to shorten the time scale forall aspects of drug discovery. Because of the ability to test largenumbers quickly and efficiently, high throughput screening (HTS) methodsare replacing conventional lead compound identification methods.

In one embodiment, high throughput screening methods involve providing alibrary containing a large number of potential therapeutic compounds(candidate compounds). Such “combinatorial chemical libraries” are thenscreened in one or more assays, as described herein to identify thoselibrary members (particular chemical species or subclasses) that displaya desired characteristic activity. The compounds thus identified canserve as conventional “lead compounds” or can themselves be used aspotential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biological synthesisby combining a number of chemical “building blocks” such as reagents.For example, a linear combinatorial chemical library such as apolypeptide (e.g., mutein) library is formed by combining a set ofchemical building blocks called amino acids in every possible way for agiven compound length (i.e., the number of amino acids in a polypeptidecompound). Millions of chemical compounds can be synthesized throughsuch combinatorial mixing of chemical building blocks. For example, onecommentator has observed that the systematic, combinatorial mixing of100 interchangeable chemical building blocks results in the theoreticalsynthesis of 100 million tetrameric compounds or 10 billion pentamericcompounds (Gallop et al. (1994) 37(9): 1233-1250).

Preparation of combinatorial chemical libraries is well known to thoseof skill in the art. Such combinatorial chemical libraries include, butare not limited to, peptide libraries (see, e.g., U.S. Pat. No.5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghtonet al. (1991) Nature, 354: 84-88). Peptide synthesis is by no means theonly approach envisioned and intended for use with the presentinvention. Other chemistries for generating chemical diversity librariescan also be used. Such chemistries include, but are not limited to:peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991), encodedpeptides (PCT Publication WO 93/20242, 14 Oct. 1993), randombio-oligomers (PCT Publication WO 92/00091, 9 Jan. 1992),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc.Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara etal. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimeticswith a Beta-D-Glucose scaffolding (Hirschmann et al., (1992) J. Amer.Chem. Soc., 114: 9217-9218), analogous organic syntheses of smallcompound libraries (Chen et al. (1994) J. Amer. Chem. Soc., 116: 2661),oligocarbamates (Cho, et al., (1993) Science, 261:1303), and/or peptidylphosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658). See,generally, Gordon et al., (1994) J. Med. Chem. 37:1385, nucleic acidlibraries (see, e.g., Strategene, Corp.), peptide nucleic acid libraries(see, e.g., U.S. Pat. No. 5,539,083) antibody libraries (see, e.g.,Vaughn et al. (1996) Nature Biotechnology, 14(3): 309-314), andPCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. (1996)Science, 274: 1520-1522, and U.S. Pat. No. 5,593,853), and small organicmolecule libraries (see, e.g., benzodiazepines, Baum (1993) C&EN,January 18, page 33, isoprenoids U.S. Pat. No. 5,569,588,thiazolidinones and metathiazanones U.S. Pat. No. 5,549,974,pyrrolidines U.S. Pat. Nos. 5,525,735 and 5,519,134, morpholinocompounds U.S. Pat. No. 5,506,337, benzodiazepines U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.).

A number of well known robotic systems have also been developed forsolution phase chemistries. These systems include, but are not limitedto, automated workstations like the automated synthesis apparatusdeveloped by Takeda Chemical Industries, LTD. (Osaka, Japan) and manyrobotic systems utilizing robotic arms (Zymate II, Zymark Corporation,Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.) which mimicthe manual synthetic operations performed by a chemist and the Venture™platform, an ultra-high-throughput synthesizer that can run between 576and 9,600 simultaneous reactions from start to finish (see AdvancedChemTech, Inc. Louisville, Ky.)). Any of the above devices are suitablefor use with the present invention. The nature and implementation ofmodifications to these devices (if any) so that they can operate asdiscussed herein will be apparent to persons skilled in the relevantart. In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

XI. High Throughput Screening

Any of the assays described herein are amenable to high-throughputscreening (HTS). Moreover, the cells utilized in the methods of thisinvention need not be contacted with a single test agent at a time. Tothe contrary, to facilitate high-throughput screening, a single cell maybe contacted by at least two, preferably by at least 5, more preferablyby at least 10, and most preferably by at least 20 test compounds. Ifthe cell scores positive, it can be subsequently tested with a subset ofthe test agents until the agents having the activity are identified.

High throughput assays for hybridization assays, immunoassays, and forvarious reporter gene products are well known to those of skill in theart. For example, multi-well fluorimeters are commercially available(e.g., from Perkin-Elmer).

In addition, high throughput screening systems are commerciallyavailable (see, e.g., Zymark Corp., Hopkinton, Mass.; Air TechnicalIndustries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.;Precision Systems, Inc., Natick, Mass., etc.). These systems typicallyautomate entire procedures including all sample and reagent pipetting,liquid dispensing, timed incubations, and final readings of themicroplate in detector(s) appropriate for the assay. These configurablesystems provide high throughput and rapid start up as well as a highdegree of flexibility and customization. The manufacturers of suchsystems provide detailed protocols the various high throughput. Thus,for example, Zymark Corp. provides technical bulletins describingscreening systems for detecting the modulation of gene transcription,ligand binding, and the like.

XII. Candidate Agent Databases.

In certain embodiments, the agents that score positively in the assaysdescribed herein (e.g. act as PPARγ agonists) can be entered into adatabase of putative and/or actual agents to inhibit, prevent, orreferse nicotine-induced pulmonary damage. The term database refers to ameans for recording and retrieving information. In certain embodimentsthe database also provides means for sorting and/or searching the storedinformation. The database can comprise any convenient media including,but not limited to, paper systems, card systems, mechanical systems,electronic systems, optical systems, magnetic systems or combinationsthereof. Typical databases include electronic (e.g. computer-based)databases. Computer systems for use in storage and manipulation ofdatabases are well known to those of skill in the art and include, butare not limited to “personal computer systems”, mainframe systems,distributed nodes on an inter- or intra-net, data or databases stored inspecialized hardware (e.g. in microchips), and the like.

XIII. Kits.

In another embodiment, this invention provides kits for the screeningprocedures and/or diagnostic procedures and/or treatment proceduresdescribed herein. Screening/diagnostic kits typically comprise one ormore reagents that specifically bind to the target that is to bescreened (e.g. PPARγ and/or PPARγ receptor).

“Therapeutic” kits typically comprise a container containing one or moremodulators of the PPARγ expression or activity and/or PPARγ receptorexpression and/or activity.

In addition, the kits optionally include labeling and/or instructionalmaterials providing directions (i.e., protocols) for the practice of themethods or use of the “therapeutics” or “prophylactics” of thisinvention. Preferred instructional materials describe the use of one oragents of this invention to inhibit, reverse, or prevent damage topulmonary tissue (e.g. from smoking or other pathologies). Theinstructional materials can also, optionally, teach preferreddosages/therapeutic regiment, counter indications and the like.

While the instructional materials typically comprise written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Mechanism of Nicotine-Induced Pulmonary FibroblastTransdifferentiation

In this example, we tested the hypothesis that in vitro nicotineexposure disrupts specific epithelial-mesenchymal paracrine signalingpathways and results in pulmonary interstitial lipofibroblast(LIF)-to-myofibroblast (MYF) transdifferentiation, resulting in alteredpulmonary development and function. Studies were done to determinewhether nicotine induces LIF-to-MYF transdifferentiation and toelucidate underlying molecular mechanism(s) involved and to determinewhether nicotine-induced LIF-to-MYF transdifferentiation could beprevented by stimulating specific alveolar interstitial fibroblastlipogenic pathway. WI38 cells, a human embryonic pulmonary fibroblastcell line, were treated with nicotine with or without specific agonistsof alveolar fibroblast lipogenic pathway, PTHrP, DBcAMP, or the potentPPAR stimulant rosiglitazone (RGZ) for 7 days. Expression of keylipogenic and myogenic markers was examined by RT-PCR, Westernhybridization, and immunohistochemistry. The effect of nicotine ontriglyceride uptake by WI38 cells and PTHrP binding to its receptor wasalso determined. Finally, the effect of transfecting WI38 cells with aPPAR expression vector on nicotine-induced LIF-to-MYFtransdifferentiation was determined. Nicotine treatment resulted insignificantly decreased expression of lipogenic and increased expressionof myogenic markers in a dose-dependent manner, indicatingnicotine-induced LIF-to-MYF transdifferentiation. This was accompaniedby decreased PTHrP receptor binding to its receptor. Thenicotine-induced LIF-to-MYF transdifferentiation was completelyprevented by concomitant treatment with PTHrP, DBcAMP, RGZ, and bytransiently overexpressing PPAR. Our data suggest nicotine inducesalveolar LIF-to-MYF transdifferentiation through a mechanism involvingdownregulation of lipogenic PTHrP-mediated, cAMP-dependent PKA signalingpathway, which can be prevented using specific molecular targets.

Materials and Methods.

Reagents

Nicotinic acetylcholine (nACh) receptor antagonists (D-tubocurarine,α-bungarotoxin, and mecamylamine) and dibutyryl cAMP (DBcAMP) wereacquired from Sigma (St. Louis, Mo.). PTHrP-(1-34) was obtained fromBachem (Torrance, Calif.), and rosiglitazone maleate (RGZ) was fromSmithKline Beecham Pharmaceuticals. nACh receptors α₃ and α₇ antibodieswere obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.). ThePPAR expression vector (pCMX-PPARγ) was kindly provided by Dr. P.Tontonoz (Univ. of California Los Angeles).

Cell Culture

The human embryonic cell line WI38 was obtained from American TypeCulture Collection (Rockville, Md.). Cells were grown in MEM plus 10%FBS at 37° C. in six-well plates, four-well slides, and 60- and 100-mmculture dishes as needed. At 70-80% confluence, the cells were treatedwith nicotine (1×10⁻⁹ or 1×10⁻⁶ M) with or without the specific agonistsof the alveolar fibroblast lipogenic pathway: PTHrP (1×10⁻⁷ M or 5×10⁻⁷M), DBcAMP (1×10⁻⁵ M or 1×10⁻⁴ M), or the potent PPAR stimulant RGZ(1×10⁻⁶ M or 1×10⁻⁵ M). Medium containing fresh chemicals was addeddaily, and at the end of 7 days, the cells were processed as needed.

Triglyceride Uptake Assay

The method used to quantitate triglyceride uptake by fetal rat lungfibroblasts has been described previously (Torday et al. (1995) BiochimBiophys Acta 1254: 198-206). Briefly, culture medium was replaced withDMEM containing 20% adult rat serum mixed with [3H]triolein (5 μCi/ml).The cells were incubated at 37° C. in 5% CO2+balance air for 4 h. At thetermination of the incubation, the medium was decanted, the cells wererinsed twice with 1 ml of ice-cold MEM, and the cells were removed fromthe culture plate after a 5- to 10-min incubation with 2 ml of a 0.05%trypsin solution. An aliquot of the cell suspension was taken forprotein assay (Bradford (1976) Anal Biochem 72: 248-254), and theremaining cell suspension was extracted for neutral lipid content.

PTHrP Receptor Binding Assay

The receptor binding assay was carried out as previously described(Torday and Rehan (2002) Am. J. Physiol. Lung Cell Mol. Physiol. 283:L130-L135; Torday and Rehan (2003) Cell Biochem Biophys 37: 235-246).The assay mixture, in a total volume of 0.1 ml, contained 50 mM Tris.HCl(pH 7.4), 2 mM dithiothreitol, 10 mM EDTA, 10 μg/ml each of proteaseinhibitors (leupeptin, pepstatin, antipain, and aprotinin), 0.5 mMphenylmethylsulfonyl fluoride, 10 mg/ml BSA, 5 mM MgCl2, 10-500 pmol[125I]-Tyr34-PTHrP-(1-34) (specific activity 1,064 Ci/mmol), 10-12 μgmembrane protein, and 1×10-10 to 1×10-6 M PTHrP-(1-34). Triplicatesamples were incubated for 30 min at 30° C. Reactions were stopped bythe addition of 0.1 ml of homogenization buffer containing 20 mg/ml ofBSA and placed in ice-cold water for 30 min, followed by centrifugationat 15,000 g for 1 min. The supernatant was aspirated, and the pellet wascounted for radioactive content with a gamma counter (model 1470;Wallace, Gaithersburg, Md.). Nonspecific binding was determined in thepresence of 1 μM nonradioactive PTHrP-(1-34). Specific binding of[¹²⁵I]-Tyr34-PTHrP-(1-34) was calculated as total binding minusnonspecific binding and expressed as femtomoles per milligrams ofprotein. Specificity of binding was determined in the presence of 1 μMPTHrP-(7-34) amide, a selective PTHrP receptor antagonist, showing thatit inhibited the binding of the radioactive ligand. In preliminarystudies, the binding was found to be linear with time for up to 60 minof incubation. The effect of nicotine (1×10⁻⁶ M) on PTHrP receptorbinding was examined without and with the nACh receptor antagonistsD-tubocurarine, α-bungarotoxin, or mecamylamine (1×10⁻⁹ to 1×10⁻⁶ M).

Isolation of Total Cellular RNA

Total RNA was isolated by lysing the cells in 4 M guanidiniumthiocyanate followed by extraction with 2 M sodium acetate (pH 4.0),phenol, and chloroform/isoamyl alcohol. RNA was precipitated withisopropanol, collected by centrifugation, vacuum dried, and thendissolved in diethylpyrocarbonate-treated water (Chomzynski and Sacchi(1987) Anal Biochem 162: 156-159). Integrity of RNA was assessed fromthe visual appearance of the ethidium bromide-stained ribosomal RNAbands following fractionation on a 1.2% (wt/vol) agarose-formaldehydegel and quantitated by absorbance at 260 nm.

Semiquantitative RT-PCR

RT-PCR probes used included PTHrP receptor: 5′-ATG TGG ATG TAG TTG CGCGTG CAG T-3′ (SEQ ID NO:1) and 3′-GGG AAG CCC AGG AAA GAT AAG GCA T-5′(SEQ ID NO:2) (445 bp); PPAR: 5′-CCC TCA TGG CAA TTG AAT GTC GTG (SEQ IDNO:3) and 3′-TCG CAG GCT CTT TAG AAA CTC CCT-5′ (SEQ ID NO:4) (757 bp);ADRP: 5′-GTT GCA GTT GAT CCA CAA CCG-3′ (SEQ ID NO:5) and 3′-TGG TAG ACAGGG ATC CCA GTC-5′ (SEQ ID NO:6) (666 bp); -smooth muscle actin (-SMA):5′-CGC AAA TAT TCT GTC TGG ATC G-3′ (SEQ ID NO:7) and 3′-TCA CAG TTG TGTGCT AGA GAC A-5′ (SEQ ID NO:8) (167 bp); nACh receptor 3: 5′-AGG CTA CAAACA CGA CAT CAA GTA-3′ (SEQ ID NO:9) and 3′-TGG CTT CTT TGA TTT CTG GTGACA-5′ (SEQ ID NO:10) (694 bp); nACh receptor 7: 5′-GGC TTC CGC GGC CTGGAC GGC GTG CAC TGT-3′ (SEQ ID NO:11) and 3′-GGC TTC CGC GGC CTG GAC GGCGTG CAC TGT-5′ (SEQ ID NO:12) (596 bp); and 18s: 5′-TTA AGC CAT GCA TGTCTA AGT AC-3′ (SEQ ID NO:13) and 3′-TGT TAT TTT TCG TCA CTA CCT CC-5′(SEQ ID NO:14) (489 bp). cDNA was synthesized from 1 μg of total RNA byRT using 100 units of SuperScript reverse transcriptase II (Invitrogen,Carlsbad, Calif.) and random primers (Invitrogen) in a 20-μl reactionmixture containing 1× SuperScript buffer (Invitrogen), 1 mM dNTP mix, 10mM dithiothreitol, and 40 units of RNase inhibitor. Total RNA and randomprimers were incubated at 65° C. for 5 min followed by 42° C. for 50min. A denaturing enzyme at 70° C. for 15 min terminated the reaction.For PCR amplification, 1 μl of cDNA was added to 25 μl of a reaction mixcontaining 0.2 μM of each primer, 0.2 mM dNTP mix, 0.5 units ofAccuPrime Taq DNA polymerase (Invitrogen), and 1× reaction buffer. PCRwas performed in a RoboCycler (Stratagene, La Jolla, Calif.). Initially,we obtained standard curves for the cycle number and the absorbanceoptical density for each of the markers examined by RT-PCR. The cyclenumber (30-38) for each PCR reaction was chosen so that the absorbanceof the amplified product was in the linear range. The PCR products werevisualized on 2% agarose gels by ethidium bromide staining, and gelswere photographed under UV lights. Band densities were quantified usingthe Eagle Eye II System (Stratagene). The expression of different mRNAswas normalized to 18s mRNA levels.

Protein Determination and Western Blot Analysis

Protein determination was made using the Bradford dye-binding method(Bradford (1976) Anal Biochem 72: 248-254). Western blotting wasperformed with modifications of methods described previously (Ayad andWong (1998) Crit Care Med 26: 1277-1282). Briefly, cells were lysedusing an extraction buffer [10 mM Tris (hydroxymethyl) aminomethane(Tris, pH 7.5), 0.25 M sucrose, 1 mM EDTA, 5 mM benzamidine, 2 mMphenylmethylsulfonyl fluoride, and 10 μg/ml each of pepstatin A,aprotinin, and leupeptin] and centrifuged at 140 g for 10 min (4° C.).Equal amounts of the protein (25 μg) from the supernatant were dissolvedin electrophoresis sample buffer and were subjected to SDS-PAGE (4-12%gradient) followed by electrophoretic transfer to a nitrocellulosemembrane. Nonspecific binding of antibody was blocked by washing withTris-buffered saline (TBS) containing 5% milk for 1 h. The blot was thensubjected to two brief washes with TBS plus 0.5% Tween 20, incubated inTBS plus 0.1% Tween 20 and the specific primary antibodies (PPAR1:2,000, Alexis Biochemicals, San Diego, Calif.; -SMA 1:50,000, Sigma;ADRP 1:3,000, a kind gift from Dr. Constantine Londos, NationalInstitute of Diabetes and Digestive and Kidney Diseases) overnight at 4°C. Blots were then washed in TBS plus 0.1% Tween 20 and then incubatedfor 1 h in secondary antibody, washed, and developed with achemiluminescent substrate (ECL; Amersham, Arlington Heights, Ill.)following the manufacturer's protocol. The densities of the specificprotein bands were quantified using a scanning densitometer (Eagle EyeII still video system, Stratagene). The blots were subsequently strippedand reprobed with anti-GAPDH (1:5,000; Chemicon, Temecula, Calif.)antibody to confirm equal loading of samples.

Transfection Protocol

For transient transfection, WI38 cells were transfected by usingLipofectamine Plus Reagent (Invitrogen). Cells were trypsinized 1 daybefore transfection and plated on 100-mm-diameter dishes so that theywere 50-80% confluent on the day of transfection. Four or 8 μg ofpCMX-PPAR cDNA were diluted in 800 μl of serum-free medium, and 20 μl ofLipofectamine Plus Reagent were added to the diluted DNA. The DNAsolution was incubated at room temperature for 15 min to precomplex DNAwith Plus Reagent. Another 30 μl of Lipofectamine Reagent diluted to 800μl in serum-free medium were combined with precomplexed DNA, and thenthe mixture incubated for 15 min at room temperature. Cells were washedwith serum-free medium twice, and then 6.4 ml of serum-free medium wereadded to each dish, followed by the addition of DNA-Lipofectamine PlusReagent complexes. The complexes were mixed into the medium gently andfurther incubated at 37° C. at 5% CO2 for 3 h. After incubation,transfection medium was replaced by complete medium containing serum andantibiotics. After incubation overnight, DNA and protein wereperiodically extracted and analyzed for DNA fragment test and Westernblot analysis. Once transfection was confirmed, the cells were treatedwith nicotine (1×10⁻⁹ M or 1×10⁻⁶ M) for 7 days, and the cell extractswere analyzed for PPAR and -SMA proteins by Western blot analysis.

Immunofluorescence Double Staining

Lipogenic and myogenic status of cultured WI38 cells was assessed bysimultaneous staining for lipid droplets and -SMA. Lipids were stainedusing oil red O staining, and -SMA expression was assessed by usinganti-SMA (cat. no. A2547, 1:1,000, mouse monoclonal IgG2, Sigma) primaryantibody. In brief, cells were cultured on Lab-Tek four-chamber slidesunder control and experimental conditions (nicotine treatment, 1×10⁻⁹ Mfor 7 days). At the end of the experimental period, slides were fixed infreshly prepared 4% paraformaldehyde. Fixed slides were washed in PBS,blocked with 3% normal goat serum (Jackson Immunoresearch Lab) in PBSfor 30 min at room temperature to block nonspecific binding, and thenincubated in primary antibody overnight at 4° C. Secondary biotinylatedanti-mouse IgG2 was used at 1:200 dilution for 30 min. The slides werethen washed 3× with PBS and with double-distilled water 2× and were thenincubated with oil red O (Sigma) for 15-30 min. Slides were rinsed 3×for 5 min and then mounted and coverslipped with Vestashield mountingmedium with 4′,6′-diamidino-2-phenylindole (Vector Laboratories)visualization under a fluorescence microscope.

Statistical Analysis

Analysis of variance for multiple comparisons with Newman-Keuls post hoctest and Student's t-test, as indicated, were used to analyze theexperimental data. P<0.05 was considered to indicate significantdifferences in the expression of lipogenic and myogenic markers amongthe control, nicotine, and nicotine plus treatment groups.

Results.

nACh Receptor Expression by WI38 Cells

Initially, we examined the expression of nACh receptors α₃ and α₇ byWI38 cells. By RT-PCR (FIG. 1A) and Western blot analysis (FIG. 1B), wefound that nACh receptors 3 and 7 were well expressed by WI38 cells.Upon nicotine stimulation (for 7 days), there were significant increasesin the expressions of both nACh receptors α₃ and α₇ (P<0.05 vs.control).

Nicotine-Induced LIF-to-MYF Transdifferentiation

Our previous studies have demonstrated that cultured developingpulmonary alveolar interstitial LIFs, exposed to stimuli that disruptfetal lung development, e.g., hyperoxia or volutrauma,transdifferentiate to MYFs via downregulation of PTHrP-mediatedcAMP-dependent PKA signaling (Rehan and Torday (2003) Cell BiochemBiophys 38: 239-250; Rizzoli et al. (1983) Endocrinology 112:1303-1312). In the present study, we examined whether cultured WI38human embryonic lung fibroblasts exposed to nicotine demonstrate asimilar effect. Furthermore, we determined whether stimulants of thePTHrP receptor-mediated cAMP-dependent PKA pathway would preventnicotine-induced alveolar LIF-to-MYF transdifferentiation.

Effect of Nicotine on mRNA Expression of Markers for the Lung FibroblastPhenotype (LIF Vs. MYF).

Exposure to nicotine (1×10⁻⁹ or 1×10⁻⁶ M) for 7 days resulted indose-dependent decreases in PTHrP receptor (−33±8% and −40±3%,respectively; means±SE), PPAR (−26±7% and −37±3%), and ADRP (−41±8% and−47±4%) mRNA expression, as determined by RT-PCR (*P<0.05 for all,nicotine vs. control; FIG. 2). This was accompanied by a concomitantdose-dependent increase in -SMA (+38±4% and +140±5%) mRNA expression(*P<0.05, nicotine vs. control).

Effect of Nicotine on Protein Expression of Markers for Lung FibroblastPhenotype (LIF Vs. MYF).

As assessed by Western blot analysis, the protein levels of PTHrPreceptor (−31±7%), PPAR (−24±2%), and ADRP (−20±3%) decreased, and thatof -SMA (+100±5%) increased significantly on exposure to nicotine(1×10⁻⁹) for 7 days (FIG. 3), indicating nicotine-induced LIF-to-MYFtransdifferentiation. This was corroborated by double-immunofluorescencestaining of WI38 cells for lipid droplets and -SMA after 7 days ofnicotine stimulation. Nicotine exposure clearly decreased lipid stainingand increased -SMA staining, the hallmarks of LIF-to-MYFtransdifferentiation (FIG. 4).

Effect of Nicotine on Triolein Uptake by WI38 Cells

To assess the effect of nicotine on LIF function, [³H]triolein uptake bycultured WI38 cells under control and experimental conditions wasmeasured. Nicotine treatment (1×10⁻⁹ M for 7 days) caused an almost 50%decrease in phenotypic triglyceride uptake (FIG. 5), which waseffectively prevented by concomitant treatment of WI38 cells with RGZ(1×10⁻⁵ M), PTHrP (5×10⁻⁷ M), or DBcAMP (1×10⁻⁴M).

Prevention of LIF-to-MYF Transdifferentiation by Stimulants ofPTHrP-Mediated, cAMP-Dependent PKA Signaling Lipogenic Pathway

The effect of specific stimulants of PTHrP-mediated, cAMP-dependent PKAlipogenic pathway on nicotine-induced LIF-to-MYF transdifferentiationwas assessed by pretreating WI38 cells with PTHrP (5×10⁻⁷ M), DBcAMP(1×10⁻⁴M), or the potent PPAR stimulant RGZ (1×10⁻⁵ M). Pretreatmentwith PTHrP, DBcAMP, or RGZ completely prevented the nicotine-induceddecreases in PTHrP receptor (FIG. 6A) and PPAR (FIG. 6B) and an increasein -SMA (FIG. 6C) protein expression, indicating prevention ofnicotine-induced LIF-to-MYF transdifferentiation. The prevention ofnicotine-induced LIF-to-MYF transdifferentiation by stimulation of thePTHrP-driven lipogenic pathway is also supported by prevention of thenicotine-induced decrease in phenotypic triglyceride uptake by thestimulants of the PTHrP-driven PKA-mediated fibroblast lipogenic pathway(FIG. 5). As PPAR expression is central to the maintenance of fibroblastlipogenic phenotype, we next examined the effect of transfection of WI38cells with PPAR expression vector on nicotine-induced LIF-to-MYFtransdifferentiation (FIG. 7). Transfected cells were treated withnicotine (1×10⁻⁹ or 1×10⁻⁶ M) for 7 days, and the expressions of PPARand -SMA were assessed by Western blot analysis. As shown in FIG. 7,under control conditions (without transfection), there was a significantdecrease in PPAR and a significant increase in -SMA protein expression.However, with PPAR transfection, these nicotine-induced changes werecompletely prevented.

Effect of Nicotine on PTHrP Binding to its Receptor

To elucidate the mechanism of nicotine-induced LIF-to-MYFtransdifferentiation, the effect of nicotine on PTHrP binding to itsreceptor was examined. Nicotine (1×10⁻⁶ M) treatment caused a 30%decrease in PTHrP binding to its receptor (fmol·90 min⁻¹·mg protein⁻¹),and this effect was prevented by pretreatment with either D-tubocurarine(1×10⁻⁶ M), a nonspecific nicotine receptor antagonist, orα-bungarotoxin, a specific α₇ nACh receptor antagonist (1×10⁻⁶ M), butnot mecamylamine, an 3 nACh receptor antagonist (FIG. 8). To determinethe functional significance of the differential effects of the α₇ and α₃nACh receptor antagonists on PTHrP binding, the effect of nicotine(1×10⁻⁶ M) on triolein uptake by WI38 cells with and withoutD-tubocurarine (1×10⁻⁹ or 1×10⁻⁶ M), -bungarotoxin (1×10⁻⁹ or 1×10⁻⁶ M),or mecamylamine (1×10⁻⁹ or 1×10⁻⁶ M) was examined. Similar to theirdifferential effects on PTHrP receptor binding, the nicotine-induceddecrease in triolein uptake was completely prevented by D-tubocurarineor α-bungarotoxin, but not by mecamylamine (FIG. 9).

Discussion.

Using the WI38 human embryonic lung fibroblast cell line as a model, wetested the hypothesis that in vitro nicotine exposure disrupts thespecific paracrine signaling pathway that results in pulmonaryLIF-to-MYF transdifferentiation and through manipulation of specificmolecular targets, this process could be prevented. First, we documentedthe expression of the specific nACh receptors α₃ and α₇ by WI38 cells,which, at least in part, have been implicated in mediatingnicotine-induced effects on developing lung structure and function(Sekhon et al. (1999) J Clin Invest 103: 637-647; Sekhon et al. (2002)Am J Respir Cell Mol Biol 26: 31-41). We, for the first time,demonstrate the presence of α₃ and α₇ nACh receptors in the culturedlung mesenchymal cells of human embryonic origin. On nicotinestimulation, the expressions of both nACh receptors α₃ and α₇ increasedsignificantly. Furthermore, the exposure of cultured WI38 cells tonicotine for 7 days resulted in significant decreases in the expressionof the key fibroblast lipogenic markers PTHrP receptor and PPAR, alongwith their downstream targets ADRP and triglyceride uptake, and anincrease in the expression of the key fibroblast myogenic marker, α-SMA.

These molecular changes were accompanied by immunohistochemical changesthat were also consistent with LIF-to-MYF transdifferentiation. Moreimportantly, the nicotine-induced molecular and functional changes werecompletely prevented by concomitant treatment with PTHrP, cAMP, or aspecific PPAR agonist, RGZ, all of which are stimulants for thefibroblast lipogenic pathway. Moreover, the nicotine effect wascompletely blocked in WI38 cells transfected with PPAR. Finally, the useof nonspecific (D-tubocurarine) and specific (α-bungarotoxin andmecamylamine) antagonists of nACh receptors suggested the specificinvolvement of the α₇ nACh receptor in the nicotine-induced decrease intriglyceride lipid uptake by the lung fibroblasts. To our knowledge,these data provide the first evidence of nicotine-induced pulmonaryalveolar LIF-to-MYF transdifferentiation and its complete prevention byconcomitant treatment with stimulants of the fibroblast lipogenicpathway. These data provide a molecular pathway for the nicotine-induceddisruption of lung development and offer an opportunity to test targetedmolecular strategies to prevent it.

Until now, there has been no specific intervention to preventnicotine-induced morbidity in the developing fetus. This is mainlybecause of failure to eliminate maternal smoking during pregnancycoupled with a lack of understanding of the molecular mechanismsinvolved in nicotine-induced morbidity (Klerman et al. (2000) TobControl 9, Suppl 3: III51-III55; Pierce and Nguyen (2002) Am. J. Respir.Cell Mol. Biol. 26: 10-13). We have recently proposed that specificdisruption of pulmonary alveolar epithelial-mesenchymal interactionsresults in interstitial LIF-to-MYF transdifferentiation, which may bethe final common pathway through which various noninflammatory andinflammatory triggers lead to chronic lung damage in the prematureinfant (Torday et al. (2003) Pediatr Pathol Mol Med 22: 189-207).Alveolar interstitial LIF-to-MYF transdifferentiation results in failedalveolarization in the developing lung, which leads to an arrest inpulmonary growth and development, the hallmarks of in uteronicotine-induced lung damage (Collins et al. (1985) Pediatr Res 19:408-412; Maritz (1988) Biol Neonate 53: 163-170; Rubin et al. (2004) DevDyn 230: 278-289; Torday et al. (2003) Pediatr Pathol Mol Med 22:189-207). Our data suggest that the likely molecular mechanisms involvedinclude decreased PTHrP binding to its receptor with resultantdownregulation of the PTHrP-stimulated cAMP-dependent PKA pathway, whichnormally induces the LIF phenotype, characterized by expression of suchlipogenic features as triglyceride accumulation and expression of PPARand ADRP. Our data indicating LIF-to-MYF transdifferentiation are alsosupported by previous observation of lower cellular lipid content in thelung tissue of both 8- and 21-day-old rat pups following in uteronicotine exposure (Maritz (1988) Biol Neonate 53: 163-170).

PTHrP is a stretch-sensitive protein expressed by the developing lungepithelium and is upregulated during late fetal lung development (Rubinet al. (2004) Dev Dyn 230: 278-289; Rubin and Torday (2000) Totowa,N.J.: Humana, p. 269-297; Torday et al. (1998) Am J Med Sci 316:205-208). It signals to the neighboring alveolar mesenchymal cellsthrough its seven-transmembrane-spanning G protein-dependent receptor,stimulating their lipogenic phenotype (Rubin et al. (1994) BiochimBiophys Acta 1223: 91-100). The critical downstream target forPTHrP/PTHrP receptor signaling is PPAR, which in turn controls otherlipogenic regulatory genes such as ADRP and leptin (Torday and Rehan(2002) Am. J. Physiol. Lung Cell Mol. Physiol. 283: L130-L135; Tordayand Rehan (2003) Cell Biochem Biophys 37: 235-246). Therefore,stimulation of PPAR induces the lipogenic phenotype, which is necessaryfor maintaining alveolar homeostasis through its autocrine effect oninterstitial fibroblasts and its paracrine effect on alveolar type IIcells (Id.). Specifically, the interstitial LIF phenotype is offunctional importance as it provides cytoprotection against oxygen freeradicals (Torday et al. (2001) Pediatr Res 49: 843-849), trafficsneutral lipid substrate to alveolar type II cells for surfactantphospholipid synthesis (Torday et al. (1995) Biochim Biophys Acta 1254:198-206), and causes alveolar type II cell proliferation (Torday et al.(2003) Pediatr Pathol Mol Med 22: 189-207). Although MYFs also seem tobe important for normal lung development, these cells are also thehallmark of chronic lung diseases in both the neonate and adult (Leslieet al. (1990) Differentiation 44: 143-149; Pache et al. (1998) ModPathol 11: 1064-1070; Toti et al. (1997) Pediatr Pulmonol 24: 22-28). Inthe developing lung, MYFs are fewer in number and are predominantlylocated at the periphery of the alveolar septa, where they very likelyparticipate in the formation of new septa (Leslie et al. (1990)Differentiation 44: 143-149; Toti et al. (1997) Pediatr Pulmonol 24:22-28). However, in chronic lung diseases, MYFs not only increase innumber but also are located in the center of the alveolar septum ingreat abundance (Toti et al. (1997) Pediatr Pulmonol 24: 22-28). In linewith these observations, using both molecular and metabolic profiling,we have previously observed that upon hyperoxic exposure, fetal rat lungLIFs transdifferentiate to MYFs (László et al. (2002) Mol Genet Metab77: 230-236; Rehan and Torday (2003) Cell Biochem Biophys 38: 239-250).Our present data also imply LIF-to-MYF transdifferentiation as thepotential underlying mechanism for the nicotine-induced lung damage inthe developing fetus.

However, the effects of in utero nicotine exposure on the developinglung are extremely complex. On the one hand, there is evidence ofenhanced functional pulmonary maturity at birth, possibly contributingto a decrease in the incidence of respiratory distress syndrome (Curetet al. (1983) Am J Obstet Gynecol 147: 446-450; Gluck and Kulovich(1973) Am J Obstet Gynecol 115: 539-546; Lieberman et al. (1992) ObstetGynecol 79: 564-570; Wuenschell et al. (1998) Am J Physiol Lung Cell MolPhysiol 274: L165-L170). In contrast, clearly, reduction in bothprenatal and postnatal lung growth occurs in children of women who smoke(Chen et al. (1987) Pediatr Pulmonol 3: 51-58; Cnattingius and Nordstrom(1996) Acta Paediatr 85: 1400-1402; Collins et al. (1985) Pediatr Res19: 408-412; Cunningham et al. (1994) Am J Epidemiol 139: 1139-1152;Gilliland et al. (2003) Am J Respir Crit Care Med 167: 917-924; Hanrahanet al. (1992) Am Rev Respir Dis 145: 1129-1135; Higgins (2002) Curr OpinObstet Gynecol 14: 145-151; Hofhuis et al. (2003) Arch Dis Child 88:1086-1090; Maritz (1988) Biol Neonate 53: 163-170; Scott (2004) TobaccoInduced Diseases 2: 3-25; Sekhon et al. (1999) J Clin Invest 103:637-647; Sekhon et al. (2001) Am J Respir Crit Care Med 164: 989-994;Walsh (1994) Hum Biol 66: 1059-1092). Significant suppression of lungalveolarization, functional residual capacity, and tidal flow volumeshas been demonstrated in the offspring of women exposed to smoke duringpregnancy. So far, the molecular mechanisms underlying these paradoxicaleffects remain largely unknown. Although acceleration of the lungdevelopmental program, including surfactant phospholipid synthesis andan increase in surfactant protein expression (Curet et al. (1983) Am JObstet Gynecol 147: 446-450′ Gluck and Kulovich (1973) Am J ObstetGynecol 115: 539-546; Lieberman et al. (1992) Obstet Gynecol 79:564-570; Wuenschell et al. (1998) Am J Physiol Lung Cell Mol Physiol274: L165-L170), have been observed to explain enhanced functionalpulmonary maturity at birth, the mechanisms underlying suppression oflung alveolarization and its functional consequences remain far lessclear. Our data provide a mechanism that explains not only failedalveolarization but also the functional pulmonary consequences followingin utero nicotine exposure, including an increased predisposition toreactive airways disease. Our data complement and extend the extensivework done by Sekhon and colleagues (Sekhon et al. (1999) J Clin Invest103: 637-647; Sekhon et al. (2001) Am J Respir Crit Care Med 164:989-994), who, using a rhesus monkey model, have reported that maternalnicotine exposure from day 26 to day 134 of gestation (term=165 days)alters fetal lung development, resulting in smaller lung volume anddecreased alveolar surface area with an accompanying increase in thesize of gas exchanging units. More importantly, concomitant with thesechanges, they reported a significant upregulation of the lung α₇ nAChreceptor and collagen I and III expression. In association with thesechanges, they also observed alterations in pulmonary function asmeasured by increased pulmonary resistance and decreased expiratoryflows (Id.). These studies, for the first time, suggested that theobserved alterations in lung mechanics in the infants of mothers whosmoke during pregnancy could be linked to the passage of nicotine acrossthe placenta, which causes increased collagen deposition and increasedairway wall dimensions in the fetal lung.

We believe a shift in lung mesenchyme phenotype from a lipogenic to amyogenic type, as predicted by our findings, not only explains theincreased collagen expression but also provides a molecular mechanismfor the altered postnatal pulmonary mechanics observed by Sekhon andcolleagues (Id.).

The complexity of the in utero effects of smoke exposure on thedeveloping lung is further suggested by the fact that even though directnicotine exposure might induce LIF-to-MYF transdifferentiation, in uterofetal smoke exposure is also accompanied by relative fetal hypoxia,which may prevent LIF-to-MYF transdifferentiation. As we have previouslydemonstrated that exposure to hyperoxia augments the spontaneouslyoccurring pulmonary LIF-to-MYF transdifferentiation, it is tempting tospeculate that the relative fetal hypoxia occurring with in utero smokeexposure may in fact have a protective effect on nicotine-inducedLIF-to-MYF transdifferentiation. The exact mechanism(s) by whichnicotine induces LIF-to-MYF transdifferentiation, in particular, thedecrease in PTHrP receptor expression and PTHrP/PTHrP receptor binding,remains to be determined. The understanding of this mechanism is likelyto be helpful designing certain specific preventive and therapeuticstrategies. However, the decreases in both PTHrP/PTHrP receptor bindingand its functional downstream effect, i.e., triolein uptake, werecompletely blocked by either tubocurarine or α-bungarotoxin, but not bymecamylamine, suggesting the specific involvement of the α₇ nAChreceptor subtype in this effect.

In summary, in addition to previously proposed mechanisms for in uteronicotine-induced lung effects, our data for the first time provideevidence for a mechanism for the direct effects of nicotine on thedeveloping mesenchyme that could permanently alter the “developmentalprogram” of the developing lung by disrupting critically importantepithelial-mesenchymal interactions. More importantly, specificinterventions that augment the pulmonary mesenchymal lipogenic pathwaycan at least partially ameliorate the very complex nicotine-induced inutero lung injury.

Example 2 In Utero Nicotine Exposure Alters Fetal Rat Lung Alveolar TypeII Cell Proliferation, Differentiation, and Metabolism

We suggested that alveolar interstitial fibroblast-to-myofibroblasttransdifferentiation may be a key mechanism underlying in uteronicotine-induced lung injury. However, the effects of in utero nicotineexposure on fetal alveolar type II (ATII) cells have not been fullydetermined. Placebo, nicotine (1 mg/kg), or nicotine (1 mg/kg)+theperoxisome proliferator-activated receptor (PPAR)-γ agonistprostaglandin J₂ (PGJ₂, 0.3 mg/kg) was administered intraperitoneallyonce daily to time-mated pregnant Sprague-Dawley rats from embryonic day6 until their death on embryonic day 20. Fetal ATII cells were isolated,and ATII cell proliferation, differentiation (surfactant synthesis), andmetabolism (metabolic profiling with the stable isotope[1,2-¹³C₂]-D-glucose) were determined after nicotine exposure in uteroor in vitro. In utero nicotine exposure significantly stimulated ATIIcell proliferation, differentiation, and metabolism. Although theeffects on ATII cell proliferation and metabolism were almost completelyprevented by concomitant treatment with PGD2, the effects on surfactantsynthesis were not. On the basis of in utero and in vitro data, weconclude that surfactant synthesis is stimulated by nicotine's directeffect on ATII cells, whereas cell proliferation and metabolism areaffected via a paracrine mechanism(s) secondary to its effects on theadepithelial fibroblasts. These data provide evidence for direct andindirect effects of in utero nicotine exposure on fetal ATII cells thatcould permanently alter the “developmental program” of the developinglung. More importantly, concomitant administration of PPAR-γ agonistscan effectively attenuate many of the effects of in utero exposure tonicotine on ATII cells.

There is compelling evidence to suggest that although maternal smokingduring pregnancy causes accelerated alveolar type II (ATII) celldifferentiation at birth, there are significant longterm deleteriouseffects on pulmonary outcome (Collins et al. 91985) Pediatr. Res. 19:408-412; Cunningham et al. 91994) Am. J. Epidemiol. 139: 1139-1152,1994; Curet et al. (1983) Am. J. Obstet. Gynecol. 147: 446-450; Gluckand Kulovich (1973) Am. J. Obstet. Gynecol. 115: 539-546; Hanrahan etal. (1992) Am. Rev. Respir. Dis. 145: 1129-1135; Lieberman et al. (1992)Obstet. Gynecol. 79: 564-570; Wuenschell et al. (1998) Am. J. PhysiolLung Cell Mol Physiol 274: L165-L170). However, the mechanism(s)underlying these paradoxical pulmonary effects remain(s) largely unknown(Pierce and Nguyen (2002) Am. J. Respir. Cell Mol. Biol. 26: 1013;Proskocil et al. (2005) Am. J. Respir. Crit. Care Med. 171: 1032-1039;Sekhon et al. (2992) Am. J. Respir. Cell Mol. Biol. 26: 31-41). ATIIcell growth and differentiation and, hence, alveolar integrity areregulated by a number of autocrine, paracrine, and endocrine factors. Inparticular, mesenchymal-epithelial interactions are critically importantfor normal lung development and injury/repair (Shannon and Hyatt (2004)Annu. Rev. Physiol. 66: 625-645; Smith and Post (1989) Am. J. Physiol.Lung Cell Mol. Physiol. 257: L174-L178; Torday et al. (2003) Pediatr.Pathol. Mol. Med., 22: 189-207; Torday et al. (2002) Am. J. Physiol.Lung Cell Mol. Physiol. 282: L405-L410 [Corrigenda. Am. J. Physiol LungCell Mol Physiol 282: April 2002, following table of contents.]). Werecently implicated the disruption of a specific epithelial-mesenchymalsignaling pathway that specifically downregulates peroxisomeproliferatoractivated receptor (PPAR)-γ expression by alveolarinterstitial fibroblasts (AIFs), resulting in AIF-to-myofibroblast (MYF)transdifferentiation in in utero nicotine exposure-induced lung injury(Rehan et al. (2005) Am. J. Physiol. Lung Cell Mol. Physiol. 289:L667-L676). It was demonstrated that augmentation of PPAR-γ in AIFs canprevent nicotine-induced AIF-to-MYF transdifferentiation. We havesuggested that this AIF-to-MYF transdifferentiation might be a keymechanism underlying the alterations in lung development following inutero nicotine exposure, explaining its long-term detrimental effects onpulmonary outcome (Id). However, the effects of in utero nicotineexposure on the pulmonary ATII cell, which secretes surfactant and isimportant to the maintenance of alveolar homeostasis, remain to be fullyelucidated. We tested the hypothesis that in utero nicotine exposuresignificantly affects ATII cell proliferation and differentiation andthat augmentation of PPAR-γ expression would reduce or prevent thenicotine-mediated alterations in ATII cell proliferation anddifferentiation. Here, we describe the effects of in utero nicotineexposure on ATII cell proliferation, differentiation, and metabolism.Our data, for the first time, help explain the mechanisms underlying theparadoxical short-term acceleration in pulmonary differentiation but apoor long-term pulmonary outcome in infants born to mothers who smokeduring pregnancy. These data provide a rationale and a molecularintervention strategy that is believed to attenuate the in uteronicotine exposure-associated effects on pulmonary outcomes.

Materials and Methods

Animals

Pathogen-free timed (embryonic day 0=day of mating) pregnantSprague-Dawley rats (200-250 g body wt) were obtained from Charles River(Hollister, Calif.) at embryonic day 3 and allowed to acclimatize untilembryonic day 6. Dams were randomized into control (placebo), nicotine,and nicotine+PPAR-γ agonist groups. Dams received placebo (diluent,normal saline), nicotine tartrate (1 mg/kg) alone, or nicotine tartrate(1 mg/kg)+the PPAR-γ agonist prostaglandin J₂ (PGJ₂, 0.3 mg/kg)intraperitoneally in 100-μl volumes once daily from embryonic day 6until they were killed with an overdose of pentobarbital sodium (200mg/kg) on embryonic day 20. The fetuses were extracted by cesareansection, and the lungs were snap frozen for later analysis or processedfor ATII cell or fibroblast culture. To determine whethernicotine-induced effects on fetal lung development specifically involvedPPAR-γ-mediated mesenchymalepithelial paracrine pathways, some animalsin the nicotine+PGJ₂ group were pretreated with a specific PPAR-γantagonist, GW-9662 (Sigma, St. Louis, Mo.; 0.25 mg/kg). All studieswere approved by the Los Angeles Biomedical Research InstituteInstitutional Review Board and were conducted in accordance with theNational Institutes of Health Guide for the Care and Use of LaboratoryAnimals.

The dose (1 mg/kg) chosen for the nicotine treatment in this study haspreviously been shown in a number of studies to result in a specificlung phenotype characterized by changes in ATII cell proliferation anddifferentiation (Maritz and Thomas (1995) Cell. Biol. Int. 19: 323-331).This dose of nicotine (0.16-1.8 mg kg body wt⁻¹ day⁻¹) is comparable tothe dose to which habitual smokers are exposed (Id.). Food and waterwere provided to the dams ad libitum, and a 12:12-h light-dark cycle wasmaintained. Three animals were used for each condition per experiment,and each experiment was repeated at least three times.

Isolation of Fetal Rat Lung ATII Cells

ATII cells were isolated using differential adherence in monolayerculture, as described previously (Battenburg et al. (1990) Biochim.Biophys. Acta 960: 441-456). Briefly, three to five dams were used perpreparation. The fetuses were delivered via cesarean section, and fetallungs were placed in Hanks' balanced salt solution without calcium andmagnesium. The lungs were chopped into small pieces with sterilescissors, the Hanks' balanced salt solution was decanted, and 5 vol of0.05% trypsin were added. A Teflon stirring bar was used to furtherdissociate the lungs by mechanical disruption of the tissue duringincubation in a 37° C. water bath. After the tissue was dispersed into aunicellular suspension, the cells were pelleted at 500 g for 10 min atroom temperature in a 50-ml polystyrene centrifuge tube. The supernatantwas decanted, and the pellet was resuspended in DMEM containing 20% FBSto yield a mixed-cell suspension of −3×10⁸ cells, as determined byCoulter particle counter (Beckman-Coulter, Hialeah, Fla.). The cellsuspension was then added to 75-cm² (T-75) culture flasks for 30-60 minto allow for differential adherence of lung fibroblasts. The unattachedcells were then transferred to another T-75 culture flask for anadditional 60 min. After this second culture period, the medium andnonadherent cells were removed from the flask and diluted with 1 vol ofculture medium. This diluted suspension was cultured overnight in a T-75culture flask at 37° C. in a CO₂ incubator to allow the ATII cells toadhere. The ATII cells were identified by their appearance in cultureunder phase contrast microscopy, lamellar body content, cytokeratinstaining, and microvillar processes. All cell cultures contained >95%ATII cells.

Cell Culture

Isolated ATII cells were cultured in DMEM+10% FBS in 6- and 96-wellplates, 100-mm dishes, and T-75 flasks, as needed, and maintained at 37°C. in a humidified incubator containing 5% CO₂-95% air. At 80-90%confluence, cells were processed for cell proliferation,differentiation, and metabolic studies (see below).

Lung Explant Culture

Explants derived from three to five litters of rats were used for eachexperiment during the course of the studies. Lungs were harvested fromfetal rats under sterile conditions. The lung tissue was chopped into1-mm cubes and incubated in 0.5 ml of Waymouth's MB-252/1 mediumcontaining penicillin (100 U/ml)-streptomycin (100 U/ml) and Fungizone(2.5 μg/ml) in six-well plates while rocking on an oscillating platform(3 cycles/min) in 5% CO2-95% air at 37° C. The explants were allowed toattach for ˜1-2 h.

Cell Proliferation

In Vivo ATII Cell Proliferation Assay.

The EnVision double-stain system (DakoCytomation, Carpentaria, Calif.)was used for immunohistochemical determination of in vivo ATII cellproliferation by double labeling with a cell proliferation-specificmarker, proliferating cell nuclear antigen (PCNA), and an ATIIcell-specific marker, surfactant protein (SP) C (SP-C). Briefly, damswere killed by cesarean section, and fetal lung tissue was fixed in 4%paraformaldehyde for 4 h. After fixation, the tissue was suspended in30% sucrose overnight, washed in PBS, and then embedded intissue-embedding medium (OCT Tissue-Tek, Sakura). Sections (8 μm) werecut using a cryotome (Leica). Endogenous peroxidase was blocked, and thesections were processed with 10 mM citrate buffer (pH 6.0) in amicrowave oven for 5 min at high power. Subsequently, sections wereincubated with the first primary antibody, mouse monoclonalproliferating cell nuclear antigen (PCNA) antibody (1:1,000 dilution;Santa Cruz Biotechnology, Santa Cruz, Calif.) for 30 min at roomtemperature; then the secondary antibody conjugated to horseradishperoxidase was added to the sections for another 30 min at roomtemperature. Vector SG (Vector Laboratories, Burlingame, Calif.) wasused as a chromogen, and blue-gray nuclear staining was consideredpositive. After the slides were washed, they were incubated withdouble-stain block, and the second primary rabbit polyclonal antibodySP-C (1:200 dilution; Chemicon, Temecula, Calif.) was applied to thesections at room temperature for 30 min and then the secondary antibodylabeled with alkaline phosphatase was applied for another 30 min. Theimmunoreaction was visualized with Vector Red (Vector Laboratories), andred cytoplasmic staining was considered positive. After dehydration, theslides were mounted with permanent mounting medium (VectaMount, VectorLaboratories). As negative controls, sections were incubated with normalserum in the absence of primary antibody. The slides were examined at×40 magnification, and ATII cells in 10 randomly selected areas (gridsize 40,000 μm²) per slide (2 slides/animal) were counted for thepurposes of statistical analysis.

Ex Vivo ATII Cell Proliferation Assay.

After in utero nicotine treatment, ATII cells were isolated as describedabove, and 5,000 cells were plated per well in 96-well plates. Accordingto the manufacturer's protocol, cell proliferation was determined by thetetrazolium dye assay, which is based on the conversion of a tetrazoliumsalt to a red formazan product by living cells (Cell ProliferationAssay, Promega).

In Vitro Cell Proliferation Assay.

Cultured ATII cells or explants were treated with nicotine under variousexperimental conditions (see below), and cell proliferation wasdetermined by Cell Proliferation Assay (Promega) or dual PCNA and SP-Clabeling, respectively (see above).

Measurement of Phospholipid Synthesis

Incorporation of [methyl-³H]choline chloride (NEN Dupont) into saturatedphosphatidylcholine was determined in monolayers of cultured explantsand ATII cells. Briefly, subconfluent monolayer cultures of ATII cellsin DMEM+0.1% FBS that had been treated with nicotine or freshly isolatedlung explant cultures in Weymouth's medium in six-well plates wereincubated with [methyl-³H]choline chloride (1 μCi/ml) for 4 h. Afterincubation, explants and cells were washed three times with ice-coldPBS. The explants and the scraped cells were thoroughly homogenized, andthe cellular lipids were extracted with chloroform-methanol (2:1) (Blighand Dyer (1959) Can. J. Biochem. Physiol., 37: 911-917). The organicphase was dried under a stream of nitrogen at 60° C., resuspended in 0.5ml of carbon tetrachloride containing 3.5 mg of osmium tetroxide, andleft at room temperature for 15 min. The reaction mixture was redriedunder nitrogen and resuspended in 70 μl of chloroform-methanol (9:1,vol/vol). The lipid extracts were transferred to silica gel plates(Kodak, Rochester, N.Y.) and developed in a chloroform-methanolwater(65:25:4) solvent system. Pure dipalmitoyl phosphatidylcholine was usedas the chromatographic standard. The developed plates were stained withbromothymol blue, blotted, and vacuum dried for 5 min at 90° C.Chromatogram spots corresponding to the migration of saturatedphosphatidylcholine were scraped from the plates and counted by liquidscintillation spectrometry. The amounts of [methyl³H]choline chlorideincorporated into saturated phosphatidylcholine were expressed asdisintegrations per minute per milligram of protein.

Western Analysis

Protein extraction and Western blot analysis for SP-B and cholinephosphate cytidylyltransferase-α (CCT-α) were performed using standardmethods. Briefly, cells were homogenized in 10 mM Tris (pH 7.5), 0.25 Msucrose, 1 mM EDTA, 5 mM benzamidine, 2 mM phenylmethylsulfonylfluoride, and 10 μg/ml each of pepstatin A, aprotinin, and leupeptin andcentrifuged at 14,000 rpm for 10 min at 4° C. Equal amounts of theprotein from the supernatant were dissolved in electrophoresis samplebuffer and subjected to SDS-polyacrylamide (4-12% gradient) gelelectrophoresis followed by electrophoretic transfer to a nitrocellulosemembrane. The membrane was blocked with 5% milk in 1×Tris-bufferedsaline containing 0.1% Tween 20 for 1 h and then incubated with rabbitanti-human SP-B polyclonal antibody (1:1,000 dilution; Chemicon)overnight at 4° C. The antibody for CCT-α (1:2,000 dilution) was a kindgift from Dr. Mallampalli (University of Iowa, Iowa City, Iowa).Subsequently, the membrane was washed with 1×Tris-buffered saline+0.1%Tween 20 and incubated with a 1:3,000 dilution of anti-rabbithorseradish peroxidase-linked whole antibody immunoglobulin G (Amersham,Arlington Heights, Ill.) for 1 h at room temperature, washed again, anddeveloped with a chemiluminescent substrate (ECL, Amersham) followingthe manufacturer's protocol. The density of the SP-B and CCT-α bands wasquantified using a scanning densitometer (Eagle Eye, Stratagene).

Stable Isotope Labeling of Intracellular Glucose Metabolites

Stable isotope labeling of intracellular glucose metabolites wasperformed according to previously described methods (Lâszló et al.(2002) Mol. Genet. Metab. 77: 230-236). Briefly, [1,2-¹³C₂]glucose waspurchased with >99% enrichment for the specified carbon positions fromIsotech (Miamisburg, Ohio). Lung ATII cells were isolated from embryonicday 20 pups from specified treatment groups and cultured in T-75 tissueculture flasks. At near confluence, cells were incubated in the presenceof DMEM containing 180 mg/dl [1,2-¹³C₂]glucose (50% isotope-enrichedglucose) for 72 h to determine the changes in carbon flux under varioustreatment conditions. The [¹³C]glucose label is readily incorporatedinto various metabolites in mammalian cells, including ribonucleic acid(through ribose synthesis), lactate (through glycolysis), glutamate(through the tricarboxylic acid cycle), and palmitate (through theformation of acetyl-CoA). As the molecular weight (atomic mass unit) ofthese molecules increases on incorporation of the heavier ¹³C atomsderived from [1,2,¹³C₂]glucose, they can be separated and quantitativelyanalyzed by gas chromatography-mass spectrometry (MS) on the basis ofchanges in their mass-to-charge ratios (m/z). This method allowssimultaneous estimation of the relative synthetic rates ofmacromolecules in response to various treatments using a commonprecursor.

Recovery of Glucose Metabolites from Lung ATII Cells

Media glucose and lactate levels were directly measured using a CobasMira chemical analyzer (Roche Diagnostics). Glucose oxidation byfibroblasts was determined on the basis of the media 13C- to 12C ratiosin released CO₂ by a Finnegan Delta-S isotope ratio mass spectroscope.The rate of ¹³CO₂ release was measured to estimate the rate of glucosecarbon oxidation by the cells, expressed as the atom percent excess,which is the proportion of ¹³C produced by the cultured cells abovebackground in calibration standard samples.

RNA ribose was isolated by acid hydrolysis (2 N HCl for 2 h) of cellularRNA after TRIzol extraction of cell pellets. Hydroxylamine in pyridineand acetic anhydride was used to derivatize ribose isolated from RNA toits aldonitrile acetate form. We monitored the ion clusters around m/z256 (carbons 1-5 of ribose, chemical ionization), m/z 217 (carbons 3-5of ribose), and m/z 242 (carbons 1-4 of ribose, electron impactionization) to detect the molar enrichment for, and the positionaldistribution of, the ¹³C label in ribose.

Lactate in the cell culture medium (0.2 ml) was extracted with ethylacetate after acidification with HCl. Lactate was derivatized to itspropylamine-heptafluorobutyric anhydrate form, and the m/z 328 ioncluster (carbons 1-3 of lactate, chemical ionization) was monitored forthe detection of M1 (recycled lactate through the pentose cycle) and M2(lactate produced by the Embden-Meyerhof-Parnas pathway) to estimatepentose cycle activity.

Fatty acids in the cell culture medium were extracted by saponificationof the TRIzol cell extract after removal of the RNA-containingsupernatant. Cell debris was treated with 30% KOH and 100% ethanolovernight, and petroleum ether was used to extract lipid. Methanolic HCl(0.5 N) was used to convert fatty acids to their methylated derivatives.Palmitate was monitored at ion cluster m/z 270. The enrichment of acetylunits and the de novo synthesis of the lipid fraction were determinedusing mass isotopomer distribution analysis for different isotopomers ofpalmitate.

Gas Chromatography-MS

Mass spectral data were obtained on an HP5973 mass-selective detectorconnected to an HP6890 gas chromatograph (GC). The settings were asfollows: GC inlet 230° C., transfer line 280° C., MS source 230° C., MSQuad 150° C. An HP-5 capillary column (30 m long, 250 μm diameter, 0.25μm film thickness) was used for glucose, ribose, and lactate analyses. ABpx70 column (25 m long, 220 μm diameter, 0.25 μm film thickness; SGE,Austin, Tex.) was used for fatty acid analysis, with specifictemperature programming for each compound studied.

Data Analysis and Statistical Methods

In vitro experiments were carried out using three cultures for eachtreatment regimen, and the experiments were repeated one to three times.Mass spectral analyses were carried out by three independent, automatedinjections of a 1-μl sample and accepted only if the sample standarddeviation was <1% of the normalized peak intensity. Statistical analyseswere performed using ANOVA. P<0.05 was considered to indicatestatistically significant differences among different treatmentconditions.

Results

Effect of Nicotine on ATII Cell Proliferation

In Vivo Assessment.

Immunohistochemical analysis by double PCNA and SP-C stainingdemonstrated an almost twofold increase in ATII cell proliferation inthe nicotine-exposed group (P<0.05, n=4; FIGS. 18A and 18B), which wascompletely blocked by the concomitant administration of the PPAR-γagonist PGJ₂. A specific PPAR-γ antagonist, GW9662, almost completelyblocked the PGJ₂ effect.

Ex Vivo Assessment.

Similar to the in vivo data, ex vivo assessment by the tetrazolium dyeassay demonstrated an almost twofold increase in cell proliferation inthe nicotine-exposed group (P<0.05, n=6) vs. the control group (FIG.18C). Concomitant treatment with PGJ₂ almost completely blocked thenicotine-induced increase in ATII proliferation. Similar to the in vivodata, GW-9662 blocked the PGJ₂ effect.

In Vitro Assessment.

Similar to the in vivo results, in lung explants in culture, 24 h ofnicotine treatment significantly increased ATII cell proliferation(P<0.05, n=4), which was blocked by PGJ₂, and, again, GW-9662 blockedthe PGJ₂ effect (FIGS. 18D and 18E). However, in contrast to the in vivoand ex vivo proliferation data, direct stimulation in vitro withnicotine for 24 h had no effect on ATII cell proliferation (FIG. 18F).

Effect of Nicotine on Phospholipid Synthesis by ATII Cells

Effect of Nicotine on [³H]Choline Incorporation into DisaturatedPhosphatidylcholine: Ex Vivo Assessment.

Surfactant phospholipid synthesis, as measured by [³H]cholineincorporation into saturated phosphatidylcholine, by the cultured ATIIcells from different experimental conditions was significantly increasedin the nicotine-exposed group vs. the control group (P<0.05, n=6; FIG.19A). However, in contrast to the proliferation data, concomitanttreatment with the PPAR-γ agonist PGJ₂, alone or in combination with thePPAR-γ antagonist GW-9662, had no effect on the nicotine-inducedincrease in phospholipid synthesis.

Effect of Nicotine on [³H]Choline Incorporation into DisaturatedPhosphatidylcholine: In Vitro Assessment.

Direct stimulation of ATII cells in vitro with nicotine for 24 h alsosignificantly increased choline incorporation (FIG. 19B). Similar to thein vivo data, concomitant treatment with PGJ₂ alone or in combinationwith the PPAR-γ antagonist GW-9662 had no effect on the nicotine-inducedincrease in phospholipid synthesis.

Effect of Nicotine on CTP:CCT-a Expression.

We also determined the protein expression of CCT-α, the rate-limitingenzyme regulating surfactant phospholipid synthesis. Matching theincrease in surfactant phospholipid synthesis, we observed a significantincrease in CCT-α protein expression after in vivo and in vitro nicotineexposures (FIGS. 20A-20B). Here again, similar to the cholineincorporation data, treatment with PGJ₂, alone or in combination withGW-9662, had no effect on the nicotine-induced increase in phospholipidsynthesis in vivo or in vitro.

Effect of Nicotine on SP-B Synthesis by ATII Cells

In vivo. Compared with the control group, in utero nicotine exposuresignificantly increased the steady-state SP-B protein level in thecultured ATII cells, as determined by Western analysis (P<0.05, n=3;FIG. 21A). Neither PPAR-γ agonist (PGJ₂) nor antagonist (GW-9662)treatment had a significant effect on this nicotine-induced increase inSP-B expression.

In vitro.

Similar to the in vivo data, direct treatment of cultured ATII cellswith nicotine resulted in a significant increase in SP-B expression thatwas unaffected by PPAR-γ agonist (PGJ₂) or antagonist (GW-9662)treatment (FIG. 21B).

Effect of in Utero Nicotine Exposure on ATII Cell Metabolism

Along with the nicotine-induced increase in ATII cell proliferation anddifferentiation, there were metabolic changes that indicated significanteffects of in utero nicotine exposure on the metabolic profile of theATII cells. Most significantly, there were changes in the pentose cyclemetabolism affecting ribonucleic acid synthesis and lipid metabolismthat may have implications for surfactant synthesis.

Ribose Synthesis.

In vivo nicotine exposure significantly altered the pentose cyclemetabolism in such a way that there was a significant increase in ribosesynthesis via the oxidative glucose-6-phosphate dehydrogenase pathway,while there was a significant decrease in ribose synthesis via thenonoxidative transketolase pathway (FIGS. 22A and 22B). These metabolicalterations were completely blocked by concomitant administration of thePPAR-γ agonist PGJ₂. In contrast, direct in vitro treatment of culturedATII cells with nicotine did not alter ribose synthesis via theoxidative or the nonoxidative pathway with or without the PPAR-γ agonistPGJ₂ (FIGS. 22C and 22D).

De Novopalmitate Synthesis.

De novo palmitate synthesis, as a function of the total palmitate in theATII cells, almost doubled on in utero exposure to nicotine (FIG. 23A).This was accompanied by a modest increase in the [¹³C]glucose labelingof the acetyl-CoA pool (FIG. 23B). Both of these changes were alsocompletely prevented by the concomitant administration of the PPAR-γagonist PGJ₂. Again, in contrast to the in vivo data, in vitrostimulation of ATII cells with nicotine for up to 72 h did not result ina significant change in de novo palmitate synthesis or [¹³C]glucoselabeling of the acetyl-CoA with or without the PPAR-γ agonist PGJ₂(FIGS. 23-C and 23D).

Discussion

The pulmonary effects of in utero nicotine exposure on the fetus areextremely complex. On the one hand, there is evidence of enhancedfunctional pulmonary maturity at birth, possibly contributing to adecrease in the incidence of respiratory distress syndrome (Curet et al.(1983) Am. J. Obstet. Gynecol. 147: 446-450; Gluck and Kulovich (1973)Am. J. Obstet. Gynecol. 115: 539-546; Lieberman et al. (1992) Obstet.Gynecol. 79: 564-570; Wuenschell et al. (1998) Am. J. Physiol Lung CellMol Physiol 274: L165-L170). On the other hand, significant reduction inprenatal and postnatal lung growth has been reported in children ofwomen who smoke (Collins et al. 91985) Pediatr. Res. 19: 408-412;Cunningham et al. 91994) Am. J. Epidemiol. 139: 1139-1152, 1994;Hanrahan et al. (1992) Am. Rev. Respir. Dis. 145: 1129-1135).Significant suppression of alveolarization, functional residualcapacity, and tidal flow volumes has been demonstrated in the offspringof nicotine-exposed pregnancies. Although the mechanisms underlying thegeneral effects of maternal smoking on fetal viability and growth aregenerally thought to be due to fetal hypoxia, the mechanisms underlyingthe seemingly paradoxical acute and chronic pulmonary effects are farmore complex and are just beginning to be elucidated (Pierce and Nguyen(2002) Am. J. Respir. Cell Mol. Biol. 26: 1013; Proskocil et al. (2005)Am. J. Respir. Crit. Care Med. 171: 1032-1039; Rehan et al. (2005) Am.J. Physiol. Lung Cell Mol. Physiol. 289: L667-L676; Sekhon et al. (2992)Am. J. Respir. Cell Mol. Biol. 26: 31-41).

The direct effects of maternal smoke on prenatal lung growth arerestricted to only those components of maternal smoke that aretransferred across the placenta. Nicotine is the major smoke constituentthat crosses the placenta and is concentrated in the fetus and, inanimal studies, has been shown to adversely affect fetal lung growth anddevelopment (Bassi et al. (1984) Pediatr. Res. 18: 127-130; Collins etal. 91985) Pediatr. Res. 19: 408-412; Luck et al. (1985) Dev. Pharmacol.Ther. 8: 384-395; Maritz and Dennis (1998) Reprod. Fertil. Dev. 10:255-261; Maritz and Thomas (1995) Cell. Biol. Int. 19: 323-331; Maritzand Woolward (1992) S. Afr. Med. J. 81: 517-519; Pastrakuljic et al.(1998) Life Sci. 63: 2333-2342; Sandberg et al. (2004) Pediatr. Res. 56:432-439; Sekhon et al. (2992) Am. J. Respir. Cell Mol. Biol. 26: 31-41).Therefore, in the human fetus as well, nicotine is likely to be themajor constituent causing pulmonary effects. We recently demonstratedthat in vitro nicotine exposure specifically disrupts parathyroidhormone-related protein-driven alveolar epithelial-mesenchymalsignaling, resulting in AIF-to-MYF transdifferentiation (Rehan et al.(2005) Am. J. Physiol. Lung Cell Mol. Physiol. 289: L667-L676). We havealso suggested that augmentation of PPAR-γ signaling, the key downstreammesenchymal target of parathyroid hormone-related protein signaling,might be a plausible intervention for prevention of nicotine-induced inutero lung damage.

Because in vitro nicotine exposure disrupts specific alveolarepithelial-mesenchymal interactions, we hypothesized that exposure tonicotine in utero, in addition to affecting ATII cell function throughits direct effects on ATII cells, would also affect it indirectly viaits effects on the AIFs. Therefore, in this study, we have specificallyexamined the effects of nicotine exposure on ATII cell proliferation,differentiation, and metabolism in vivo as well as in vitro;furthermore, the effect of concomitant administration of the PPAR-yagonist PGJ₂ was also examined.

We found that in utero fetal exposure to nicotine through parenteraladministration to the mother significantly increased ATII cellproliferation and surfactant synthesis and altered glucose and lipidmetabolism. In vivo and in vitro data suggest that surfactant synthesisis stimulated via nicotine's direct effects on ATII cells, whereas cellproliferation and metabolism are affected via the paracrine mechanismsecondary to its effects on the adepithelial fibroblasts, suggestingdirect and indirect effects of in utero nicotine exposure on fetalpulmonary ATII cells. These paracrine effects were almost completelyprevented by the concomitant administration of the PPAR-γ agonist PGJ₂.

Our observation of increased ATII cell proliferation in response to inutero nicotine exposure is consistent with the observations of Maritzand Thomas (Maritz and Thomas (1995) Cell. Biol. Int. 19: 323-331). Wehave extended their observations by demonstrating that ATII cellproliferation increased with in vivo nicotine exposure, but not withdirect in vitro nicotine stimulation of ATII cells, suggesting a likelyparacrine mechanism underlying this response. Furthermore, the in vivonicotine-induced ATII cell proliferation was, to a large extent,prevented by the concomitant administration of the PPAR-γ agonist PGJ₂.The possibility that the nuclear transcription factor PPAR-γ is playinga role in this response is further demonstrated by the observation thatthe PPAR-γ agonist-mediated prevention of the increase in ATII cellproliferation was completely blocked by the PPAR-γ-specific antagonistGW-9662. This finding is consistent with the antimitogenic role ofPPAR-γ in other systems, where it has been shown that PPAR-γ can inhibitcell proliferation by regulating the activation of cyclins andcycin-dependent kinases (Dubey et al. (1993) Am. J. Physiol. Regul.Integr. Comp. Physiol. 265: R726-R732; Law et al. (2000) Circulation101: 1311-1318; Wakino et al. (2000) J. Biol. Chem. 275: 22435-22441).Our recent work has clearly demonstrated that AIFs that are locatedadjacent to ATII cells express PPAR-γ (Rehan et al. (2006) Exp. LungRes. 32: 379-393) and nicotine treatment downregulates PPAR-γ expressionby these fibroblasts (Rehan et al. (2005) Am. J. Physiol. Lung Cell Mol.Physiol. 289: L667-L676), which, in turn, may disturb the balance offibroblast-derived epithelial cell growth-stimulatory and -inhibitoryparacrine mediators, resulting in ATII cell proliferation.

With regard to nicotine's effect on surfactant synthesis, although alarge body of work has been generated on the effects of cigarette smokeon the surfactant system in the adult, there is very limited informationon the effects of nicotine exposure in the developing lung in utero.Lieberman et al. Lieberman et al. (1992) Obstet. Gynecol. 79: 564-570,reported higher amniotic fluid-saturated phosphatidylcholine contents inhuman fetuses exposed to intrauterine smoke. A related study reported anincrease (Maritz and Thomas (1995) Cell. Biol. Int. 19: 323-331) in thelamellar body content of pulmonary ATII cells after intrauterinenicotine exposure. Recently, however, Chen et al. (2005) Pediatr.Pulmonol. 39: 97-102, did not find a significant difference in thesaturated phosphatidylcholine contents in the lung tissue ofnicotine-exposed vs. nonexposed rat pups on postnatal day 1. Incontrast, after in utero nicotine exposure, we found an increase insaturated phosphatidylcholine synthesis, as measured by cholineincorporation ex vivo by the fetal rat lung explants. Similarly, thereis conflicting information on the effects of nicotine on SP expressionby the developing lung. Chen et al. (Id.) did not find any effect of inutero nicotine exposure (from day 3 to day 21 of gestation) on the lungmRNA expression of SP-A, -B, -C, and -D in the newborn rat. However,Wuenschell et al. (1998) Am. J. Physiol Lung Cell Mol Physiol 274:L165-L170, reported significant increases in the expression of SP-A and-C mRNAs in a murine developing lung explant model. Hermans et al.(2001) Pediatr. Res. 50: 487-494, did not find any significantdifferences in amniotic fluid SP-A levels at full term in smoke-exposedvs. non-smoke-exposed pregnancies. The conflicting results in thesestudies are likely to be related to differences in the models used(species and stage of lung development), duration of nicotine exposure(acute vs. chronic), whether smoke or nicotine was used as a challenge,and the end points, for example, whether phospholipid synthesis,secretion, or total pool size was examined.

The effects of in utero nicotine exposure on glucose metabolism in fetalrat ATII cells have been only sparingly studied. In the whole lung of14-day-old suckling pups, after nicotine exposure during pregnancy andlactation, glucose turnover was increased, and glycolysis andglycogenolysis were decreased (Kordom et al. (2003) Exp. Lung Res. 29:79-89). This was attributed to an inhibition of the activity ofphosphofructokinase. We found that although in vivo nicotine exposuresignificantly increased ribose synthesis via the oxidativeglucose-6-phosphate dehydrogenase pathway and decreased it via thenonoxidative transketolase pathway, direct in vitro treatment ofcultured ATII cells with nicotine did not alter ribose synthesis via theoxidative or the nonoxidative pathway. This discrepancy between in vitroand in vivo effects of nicotine on ATII cell glucose metabolism issimilar to the previously reported discrepancy in the effect of nicotineexposure on phosphofructokinase activity in adult lung tissue under invivo and in vitro conditions (Id). Since this effect was observed onlyunder in vivo nicotine exposure conditions and not on direct in vitronicotine stimulation of ATII cells, it is also likely to be mediated viaa paracrine mechanism. Since ATII cells normally depend on the adjoininglipid-laden AIFs for their supply of neutral lipids for surfactantphospholipidsynthesis (Torday et al. (2002) Am. J. Physiol. Lung CellMol. Physiol. 283: L130-L135), the loss of lipogenic potential of theseAIFs in response to in vivo nicotine exposure might be a trigger for theincrease in de novo palmitate synthesis by the ATII cells under in vivoconditions. Since the nicotine-induced in vivo alterations in ATII cellglucose and lipid metabolisms were blocked by the concomitantadministration of the PPAR-y agonist PGD2, downregulation of PPAR-γ inAIFs is very likely the key modulator for these metabolic changes.

Taken together, these data indicate that in utero nicotine exposuresignificantly affects ATII cell proliferation, differentiation, andmetabolism via direct and indirect effects of nicotine on ATII cells.The stimulation of ATII cell proliferation and surfactant synthesisafter in utero nicotine exposure likely explains the decrease in theincidence of respiratory distress syndrome in infants of mothers whosmoke during pregnancy (Curet et al. (1983) Am. J. Obstet. Gynecol. 147:446-450; Gluck and Kulovich (1973) Am. J. Obstet. Gynecol. 115: 539-546;Lieberman et al. (1992) Obstet. Gynecol. 79: 564-570; Wuenschell et al.(1998) Am. J. Physiol Lung Cell Mol Physiol 274: L165-L170). However,since nicotine also disrupts the homeostatic alveolarepithelialmesenchymal interactions, resulting in AIF-to-MYFtrans-differentiation (Rehan et al. (2005) Am. J. Physiol. Lung CellMol. Physiol. 289: L667-L676), the stimulatory effect of in uteronicotine exposure on ATII surfactant synthesis ultimately fails. Thisprobably also explains why Chen et al. (supra) observed a significantdecrease in the saturated phosphatidylcholine content of the lung tissuein nicotine-exposed vs. nonexposed rat pups on postnatal days 35 and 42,even when they found no differences between the two groups on postnatalday 1. Therefore, it is likely that, after in utero nicotine exposure,the combination of a nicotine-induced AIF-to-MYF transdifferentiationand a decrease in surfactant synthesis, in the long run, impacts lungfunction adversely.

Although the mechanisms underlying the effects of in utero nicotineexposure on ATII cell proliferation and differentiation remain to befully elucidated, it seems that surfactant synthesis is stimulated vianicotine's direct effects on ATII cells, whereas cell proliferation andmetabolism are affected via a paracrine mechanism secondary to itseffects on the adepithelial fibroblasts.

However, the key finding that PPAR-γ agonist administration almostcompletely blocked the nicotine-induced effects on ATII cellproliferation and differentiation indicates that manipulation of PPAR-γexpression can at least partially prevent or even reverse thenicotine-induced ATII cell effects. Our previous finding that directtreatment of AIFs with nicotine downregulates PPAR-γ expression (Rehanet al. (2005) Am. J. Physiol. Lung Cell Mol. Physiol. 289: L667-L676) isconsistent with our present findings and the proposed paracrinemechanism for the observed effects on ATII cell proliferation anddifferentiation. It is also important to note that ATII cells and AIFsexpress nicotinic acetylcholine receptors, which have been shown to beupregulated upon nicotine stimulation.

In summary, in addition to previously proposed mechanisms for in uteronicotine-induced lung effects, our data, for the first time, provideevidence for direct and indirect effects of in utero nicotine exposureon fetal pulmonary ATII cells that could permanently alter the“developmental program” of the developing lung. The use of PPAR-γagonists can, at least partially, ameliorate the complexnicotine-induced lung injury in utero. In this regard, it is importantto note that to more effectively prevent the maternal nicotineexposure-induced effects on the offspring's ATII cell proliferation,differentiation, and metabolism, PPAR-γ agonist intervention might beneeded not only during gestation but also during lactation.

Example 3 Nicotine-Induced Lung Injury can be Reversed

The data presented below indicate that using the approach outlinedabove, the nicotine-induced lung injury can not only be prevented butalso reversed. It is believed this is first demonstration of thepossibility of a reversal of the nicotine-induced lung injury by usingany approach. As illustrated below, in cultured human lung fibroblasts,we first documented the nicotine-induced changes in fibroblast phenotypeto a muscle like phenotype and then by molecularly manipulating thesenicotine treated fibroblasts, we were able the reverse thenicotine-induced fibroblast phenotype to its original non-nicotineexposed phenotype.

Embryonic human lung fibroblasts initially exposed to nicotine (10⁻⁹M)for 7 days and then treated with PPARγ agonists (RGZ, PTHrP, or cAMP)for the following 7 days. Even after nicotine treatment was stopped,PTHrP receptor expression continued to be significantly lower innicotine treated controls compared to untreated controls. In contrast,treatment with RGZ, PTHrP, or cAMP not only reversed nicotine-induceddecrease in PTHrP receptor expression, in fact, even markedly increasedin comparison to untreated controls.

Similar to the reversal of PTHrP receptor expression, nicotine-induceddecrease in PPARγ expression was also reversed with treatments with RGZand PTHrP. Although with cAMP there was a trend towards an increase inPPARγ expression, it did not reach statistical significance.

αSMA expression was significantly higher in nicotine treated controlsvs. untreated controls even after 7 days after stopping nicotinetreatment. However, treatment with RGZ, PTHrP, or cAMP reversed thenicotine-induced increase in αSMA expression.

To assess the functional relevance of the reversal of thenicotine-induced fibroblast phenotype, we examined the triolein uptake(a functional marker of normal lung fibroblast phenotype) followingdifferent experimental conditions. As predicted, nicotine treatmentresulted in a significant decrease in triolein uptake, which was atleast partially blocked by treatments with all 3 (RGZ, PTHrP, and cAMP)agonists of the normal fibroblast phenotype.PTHrP signaling pathway.

Example 4 Reversal of Nicotine-Induced AlveolarLipofibroblast-to-Myofibroblast Transdifferentiation by Stimulants ofParathyroid Hormone-Related Protein Signaling

Nicotine exposure disrupts the parathyroid hormone-related protein(PTHrP)-driven alveolar epithelial-mesenchymal paracrine-signalingpathway, resulting in the transdifferentiation of pulmonarylipofibroblasts (LIFs) to myofibroblasts (MYFs), which seems to becentral to altered pulmonary development and function in infants born tomothers who smoke during pregnancy. Modulation of PTHrP-driven signalingcan almost completely prevent nicotine-induced LIF-to-MYFtransdifferentiation. However, once this process has occurred, whetherit can be reversed is not known. Our objective was to determine ifnicotine-induced LIF-to-MYF transdifferentiation could be reversed byspecifically targeting the PTHrP-mediated alveolarepithelial-mesenchymal paracrine signaling. WI38 cells, a humanembryonic pulmonary fibroblast cell line, were initially treated withnicotine for 7 days and LIF-to-MYF transdifferentiation was confirmed bydetermining the downregulation of the key lipogenic marker, peroxisomeproliferator-activated receptor γ (PPARγ) and upregulation of the keymyogenic marker, α-smooth muscle actin (αSMA). Because downregulation ofthe PPARγ signaling pathway is the key determinant of LIF-to-MYFtransdifferentiation, cells were treated with three agonists of thispathway, PTHrP, dibutryl cAM3 (DBcAMP), or rosiglitazone (RGZ) for 7days, and the expression of the PTHrP receptor, PPARγ, αSMA, andcalponin was determined by Western analysis and immunohistochemistry.Simultaneously, fibroblast function was characterized by measuring theircapacity to take up triglycerides. Nicotine-induced LIF-to-MYFtransdifferentiation was almost completely reversed by treatment withRGZ, PTHrP, or DBcAMP, as determined by protein and functional assays.Using a specific molecular approach and targeting specific molecularintermediates in the PTHrP signaling pathway, to our knowledge, this forthe first time, demonstrates the reversibility of nicotine-inducedLIF-to-MYF transdifferentiation, suggesting not only the possibility ofprevention but also the potential for reversal of nicotine-induced lunginjury.

Introduction

There is strong epidemiologic and experimental evidence that fetalexposure to maternal smoking during gestation results in detrimentallong-term effects on lung growth and function (Maritz (1988) Biol.Neonate 53: 163-170; Walsh (1994) Hum. Biol. 66: 1059-1092; Chen et al.(1987) Pediatr. Pulmonol. 3: 51-58; Collins et al. (1985) Pediatr. Res.19: 408-412; Cunningham et al. (1994) Am. J. Epidemiol. 139: 1139-1152).Significant suppression of alveolarization, functional residualcapacity, and tidal volume has been demonstrated in the offspring ofnicotine-exposed pregnancies (Maritz (1988) Biol. Neonate 53: 163-170;Walsh (1994) Hum. Biol. 66: 1059-1092; Chen et al. (1987) Pediatr.Pulmonol. 3: 51-58; Collins et al. (1985) Pediatr. Res. 19: 408-412;Cunningham et al. (1994) Am. J. Epidemiol. 139: 1139-1152; Hanrahan etal. (1992) Am. Rev. Respir. Dis. 145: 1129-1135; Sekhon et al. (2002)Am.J. Respir. Cell. Mol. Biol. 26: 31-41). Although the molecularmechanisms underlying the long-term pulmonary effects following in uteronicotine exposure remain poorly understood (Pierce and Nguyen (2002) Am.J. Respir. Cell. Mol. Biol. 26: 10-13; Rehan et al. (2005) Am. J.Physiol. Lung Cell Mol. Physiol. 289: L667-L676), our recent work hasclearly implicated the disruption of the homeostatic alveolarepithelial-mesenchymal parathyroid hormone-related protein (PTHrP)paracrine-signaling pathway following in utero nicotine exposure thatresults in the transdifferentiation of pulmonary lipofibroblasts (LIFs)to myofibroblasts (MYFs) (Rehan et al. (2005) Am. J. Physiol. Lung CellMol. Physiol. 289: L667-L676). Normally, under the influence of cyclicstretch, e.g., during normal breathing, PTHrP is secreted by thealveolar type II cell, which binds to its cognate receptor on thelipofibroblast, activating the cAMP-dependent PKA-mediated lipogenicpathway (FIG. 24) and upregulating PPARc and its downstream targets,ADRP, which facilitates triglyceride uptake by the lipofibroblast(Schultz et al. (2002) Am. J. Physiol. Lung Cell Mol. Physiol. 283:L288-L296) and leptin, which stimulates surfactant phospholipid andprotein synthesis by the alveolar type II cell (Torday et al. (2002) Am.J. Physiol. Lung Cell Mol. Physiol. 282: L405-L410, erratum in Am. J.Physiol. Lung Cell Mol. Physiol. (2002) 282(4) Section L). Thetriglycerides taken up by the lipofibroblast are then trafficked to theATII cell as substrate for surfactant phospholipid synthesis (Torday etal. (1995) Biochem. Biophys. Acta 1254: 198-206). In fact, forsurfactant phospholipid synthesis, ATII cells are essentially dependenton lipofibroblasts to recruit neutral lipids from the circulation to betrafficked to the ATII cells and incorporated into surfactantphospholipids. Nicotine exposure downregulates the PTHrP signalingpathway resulting in LIF-to-MYF transdifferentiation, which seems to becentral to altered pulmonary development and function in infants born tomothers who smoke during pregnancy. Even more important, we have shownthat PTHrP signaling pathway agonists can almost completely preventnicotine-induced LIF-to-MYF transdifferentiation (FIG. 24).

The present series of experiments was designed to determine whether, byspecifically targeting the PTHrP-mediated alveolarepithelial-mesenchymal paracrine-signaling pathway, nicotine-inducedLIF-to-MYF transdifferentiation can be reversed after it has occurred.The data presented herein clearly suggest that using a specificmolecular approach that targets the intermediates in the PTHrP signalingpathway, nicotine-induced LIF-to-MYF transdifferentiation can bereversed, suggesting not only the possibility of prevention but also thepotential for reversal of nicotine-induced lung injury. This clearly hassignificant potential therapeutic implications for both in utero andpostnatal nicotine-induced lung injury.

Materials and Methods

Reagents

PTHrP-(1-34) was obtained from Bachem (Torrance, Calif.) androsiglitazone from Cayman Chemical (Ann Arbor, Mich.). DBcAMP and alphasmooth muscle actin (cSMA) antibody were obtained from SigmaBiochemicals (St. Louis, Mo.). Calponin antibody was obtained from SantaCruz Biotechnology, Inc. (Santa Cruz, Calif.), peroxisomeproliferator-activated receptor γ (PPARγ) antibody from AlexisBiochemicals (San Diego, Calif.), and adipocyte differentiation relatedprotein (ADRP) antibody was a kind gift from Dr. Constantine Londos,NIDDK.

Cell Culture

The human embryonic cell line WI38 was obtained from the American TypeCulture Collection (Rockville, Md.). Cells were grown in minimumessential medium (MEM)+10% fetal bovine serum at 37° C. in six-wellplates, two well slides, and 60-mm and 100-mm culture dishes, as needed.At 70%-80% confluence, cells were initially treated with nicotine(1×10⁻⁹ M) for 7 days and LIF- to MYF transdifferentiation was confirmedby determining the downregulation of the key lipogenic marker, PPARγ,and upregulation of the key myogenic marker, αSMA, by Western analysis.Subsequently, the cells were treated with rosiglitazone (RGZ) (1×10⁻⁵ M)(a PPARγ agonist), PTHrP (5×10⁻⁷ M), or DBcAMP (1×10⁻⁵ M) for 7 days,and the expression of PTHrP receptor, PPARγ, ADRP, calponin, and cSMAwas determined by Western analysis and immunohistochemistry.Simultaneously, fibroblast function was characterized by measuring theircapacity to take up triglycerides. Some experiments were performed inthe presence of a specific PPARγ antagonist, GW9662. Medium containingfresh chemicals was added daily, and at the end of the experimentalperiod the cells were processed as needed.

Triglyceride Uptake Assay

The method used to quantitate triglyceride uptake by fetal rat lungfibroblasts has been described previously (Torday et al. (1995) Biochem.Biophys. Acta 1254: 198-206). Briefly, culture medium was replaced withDMEM containing 20% adult rat serum mixed with [³H]triolein (5 μCi/ml).The cells were incubated at 37° C. in 5% CO₂-air balance for 4 h. At thetermination of the incubation, the medium was decanted, the cells wererinsed twice with 1 ml of ice-cold phosphate buffered saline (PBS), andthe cells were removed from the culture plate after a 5-10 minincubation with 2 ml of a 0.05% trypsin solution. An aliquot of the cellsuspension was taken for protein assay, and the remaining cellsuspension was extracted for neutral lipid content.

Protein Determination and Western Blot Analysis

Protein determination was made using the Bradford dye-binding method(Bradford (1976) Anal. Biochem. 72: 248-254). For Western blotting,cells were lysed using an extraction buffer [10 mM tris (hydroxymethyl)aminomethane (Tris, pH 7.5), 0.25 M sucrose, 1 mM EDTA, 5 mMbenzamidine, 2 mM phenylmethylsulfonyl fluoride, and 10 (j.tg/ml each ofpepstatin A, aprotinin, and leupeptin], and centrifuged at 140 g for 10min (4° C.). Equal amounts of the protein (25 μg) from the supernatantwere dissolved in electrophoresis sample buffer and were subjected tosodium dodecyl sulfate-polyacrylamide (4%-12% gradient) gelelectrophoresis (SDS-PAGE) followed by electrophoretic transfer to anitrocellulose memane. The nonspecific binding of antibody was blockedby washing with Tris-buffered saline (TBS) containing 0.1% Tween 20(TBST) and 5% milk for 1 h. The blot was then subjected to two briefwashes with TBST plus 0.5% Tween 20, incubated in TBST plus 0.1% Tween20 and the specific primary antibodies (PPARγ 1:2000, AlexisBiochemicals, San Diego, Calif.; cSMA 1:50,000, Sigma, St. Louis, Mo.:ADRP 1:3000) overnight at 4° C. Blots were then washed in TBST plus 0.1%Tween 20 and then incubated for 1 h in secondary antibody, washed, anddeveloped with a chemiluminescent substrate [enhanced chemiluminescence(ECL); Amersham, Arlington Heights, Ill.] following the manufacturer'sprotocol. The densities of the specific protein bands were quantifiedusing a scanning densitometer (Eagle Eye II still video system,Stratagene, La Jolla, Calif.). The blots were subsequently stripped andreprobed with anti-GAPDH (1:5000, Chemicon, Inc., Temecula, Calif.)antibody to confirm equal loading of samples.

Immunofluorescence Double Staining

The lipogenic and myogenic status of cultured WI38 cells was assessed bysimultaneous staining for either lipid droplets or ADRP and αSMA. Lipidswere detected by using either oil red O staining or ADRP expression(polyclonal anti-ADRP 1:500), and αSMA expression was assessed by usinganti-αSMA (1:1000, mouse monoclonal IgG_(2a), Sigma, catalog No. A2547)primary antibody. In brief, cells were cultured on Lab-Tek® 2-chamberslides under control and experimental conditions (nicotine treatment,1×10⁻⁹ M for 7 days). At the end of the experimental period, slides werefixed in freshly prepared 4% paraformaldehyde. Fixed slides were washedin PBS, blocked with 3% normal goat serum (Jackson Immunoresearch Lab)in PBS for 30 min at room temperature to block nonspecific binding, andthen incubated for 1 h at room temperature with primary antibodies αSMAand ADRP. Thereafter, slides were washed in PBS for 5 min, thenincubated in the dark for 30 min using a mixture of secondary goatanti-mouse IgG_(2a)-conjugated FITC and goat anti-rabbit IgG conjugatedTexas red. The slides were then washed in PBS for 5 min and mountedusing Vesta shield mounting medium with DAPI (Vector Laboratories,Burlingame, Calif.). For αSMA and oil red O double staining, the slideswere incubated with cSMA for 1 h at room temperature, followed bysecondary goat anti-mouse IgG_(2a) with FITC for 30 min. The slides werewashed in distilled water and then incubated with oil red O (Sigma, St.Louis, Mo.) for 15 min. Slides were rinsed three times for 5 min andthen mounted and visualized under a fluorescence microscope.

Statistical Analysis

Analysis of variance for multiple comparisons using the Newman-Keulspost hoc test was used to analyze the experimental data. A p value lessthan 0.05 was considered to indicate significant differences amongvarious experimental groups.

Results

Nicotine Induces Alveolar Interstitial Lipo- to MyofibroblastTransdifferentiation

In accord with our previous observations [9], by Western analysis wefirst confirmed that nicotine treatment of cultured embryonic human lungfibroblasts (WI38 cells) for 7 days resulted in significant decreases inthe expression of PTHrP receptor (*p<0.001 vs. control; n=3), PPARγ(*p<0.001 vs. control; n=3), and ADRP (*p<0.05 vs. control; n=3), andsignificant increases in the expression of αSMA (*p<0.001 vs. control;n=3) and calponin (*p<0.001 vs. control; n=3) (FIG. 25).

Reversal of Nicotine-Induced Alveolar Interstitial Lipoto-MyofibroblastTransdifferentiation by PTHrP Signaling Pathway Agonists

Following 7 days exposure to nicotine (10⁻⁹M), embryonic human lungfibroblasts were kept in culture either without (7d nicotine-onlytreatment group) or with PTHrP signaling agonists [RGZ (1×10⁻⁵ M), PTHrP(5·×10⁻⁷ M), or DBcAMP (1×100⁻⁵ M)] for the following 7 days.Subsequently, expression of the various markers for the fibroblastphenotypes (PTHrP receptor, PPARγ, ADRP, αSMA, and calponin) wasevaluated by Western analysis. In the 7d nicotine-only treatment group,despite the absence of continued nicotine exposure, the expression ofPTHrP receptor, PPARγ, and ADRP continued to be significantly lower andthe expression of αSMA and calponin significantly higher compared tountreated controls. In contrast, however, treatment with PTHrP pathwayagonists almost completely reversed the nicotine-induced changes in theexpression of PTHrP receptor (*p<0.05 vs. control and #p<0.001 vs.nicotine; n=3; FIG. 26A), PPARγ (*p<0.01 vs. control and ^(#)p<0.05 vs.nicotine; n=3; FIG. 26B), ADRP (*p<0.01 vs. control and ^(#)p<0.001 vs.nicotine; n=3; FIG. 26C), cSMA (*p<0.01 vs. control and ^(#)p<0.001 vs.nicotine; n=3; FIG. 27A) and calponin (*p<0.001 vs. control and^(#)p<0.001 vs. nicotine; n=3; FIG. 27B). This reversal ofnicotine-induced LIF-to-MYF transdifferentiation was corroborated byimmunochemistry (FIG. 28).

Immunofluorescence costaining for ADRP and αSMA or lipid droplets (oilred O) and cSMA following nicotine treatment resulted in markeddecreases in ADRP and lipid droplet staining and a marked increase inαSMA staining. Both RGZ and cAMP treatments reversed thenicotine-induced decreases in ADRP and lipid droplet staining and theincrease in αSMA staining.

Time Frame for PTHrP Signaling Pathway Agonist-Mediated Reversal ofNicotine-Induced Lipo-to-Myofibroblast Transdifferentiation

In the above experiments reversal of nicotine-induced molecular changeswas assessed following 7 days of treatment with PTHrP signaling pathwayagonists. To assess how soon this reversal might occur, we next examinedthe evidence for the reversal of the nicotine-induced LIF-to-MYFtransdifferentiation following only 24 h of treatment with PTHrPsignaling pathway agonists (FIG. 29). Similar to the 7-day data, therewas clear evidence of reversal of nicotine-induced LIF-to-MYFtransdifferentiation even after only 24 h of treatment with PTHrPsignaling agonists.

Functional Relevance of Reversal of Nicotine-MediatedLipo-to-Myofibroblast Transdifferentiation

To determine whether reversal of nicotine-induced LIF-to-MYFtransdifferentiation by stimulants of PTHrP signaling was functionallyrelevant, we next examined triglyceride uptake by fibroblasts followingvarious treatment conditions. Robust triglyceride uptake is a functionalcharacteristic of LIFs and is not a prominent feature of MYFs. Aspredicted, nicotine treatment resulted in a significant decrease intriolein uptake, which was at least partially blocked by treatment withall three agonists of the PTHrP signaling pathway (*p<0.001 vs. controland ^(#)p<0.001 vs. nicotine groups, n=3; FIG. 30).

PPARy Expression is Central to the Reversal of Nicotine-InducedLipo-to-Myofibroblast Transdifferentiation by Stimulants of the PTHrPSignaling Pathway

Because our work has demonstrated that PPARγ expression is central tothe maintenance of the alveolar interstitial fibroblast lipogenicphenotype, we next examined the centrality of PPARγ expression in thereversal of nicotine-induced LIF-to-MYF transdifferentiation by PTHrPsignaling agonists. As expected, pretreatment with a specific PPARγantagonist, GW9662, completely blocked the molecular protection againstnicotine-induced LIF-to-MYF transdifferentiation by all three PTHrPsignaling pathway agonists (PTHrP, DBcAMP, and RGZ) studied, suggestingthe specificity of PPARγ activation in the protection provided by PPARγagainst nicotine-induced LIF-to-MYF transdifferentiation (*p<0.001 vs.control; ^(#)p<0.01 vs. nicotine; ̂p<0.001 vs. nicotine+RGZ; ^(@)p<0.001vs. nicotine+PTHrP; and ^($)p<0.001 vs. nicotine+cAMP groups; n=3; FIG.31).

Discussion

Nicotine disrupts specific PTHrP-driven alveolar epithelial-mesenchymalparacrine signaling, resulting in the trans-differentiation of pulmonaryLIFs to MYFs. We have suggested that LIF-to-MYF transdifferentiation isa central mechanism that contributes to the altered pulmonarydevelopment and function in infants born to mothers who smoke duringpregnancy (Rehan et al. (2005) Am. J. Physiol. Lung Cell Mol. Physiol.289: L667-L676). Nicotine exposure downregulates the PTHrP-drivencAMP-dependent protein kinase A pathway, which normally induces thealveolar interstitial fibroblast lipogenic phenotype that is essentialfor normal pulmonary development and homeostasis (Rubin et al. (2004)Dev. Dyn. 230: 278-289; Torday et al. (2003) Pediatr. Pathol. Mol. Med.22: 189-207). LIF-to-MYF transdifferentiation results in failedalveolarization in the developing lung, which leads to an arrest inpulmonary growth and development, the hallmarks of in uteronicotine-induced lung damage (Collins et al. (1985) Pediatr. Res. 19:408-412; Maritz and Dennis (1998) Reprod. Fertil. Dev. 10: 255-261).Furthermore, modulation of the fibroblast PTHrP-driven signaling pathwaycan almost completely “prevent” nicotine-induced LIF-to-MYFtransdifferentiation (Rehan et al. (2005) Am. J. Physiol. Lung Cell Mol.Physiol. 289: L667-L676). However, once this process (LIF-to-MYFtransdifferentiation) has occurred, whether it could be reversed was notknown before this study. In this experiment, consistent with ourprevious observations, we initially confirmed that 7-day treatment ofWI38 cells with nicotine (1×10⁻⁹) resulted in significant decreases inPTHrP receptor, PPARy, and ADRP protein expression and significantincreases in SMA and calponin protein expression, thereby confirmingeither 24 h or 7 days reversed nicotine-induced LIF-to-MYFtransdifferentiation, as demonstrated by Western analysis,immunohistochemistry, and triglyceride uptake.

Smoking is the most common addiction among pregnant women in the U.S. Anestimated 12% of pregnant women are reported to smoke during pregnancy,and despite all the publicity on the harmful effects of smoking on thepregnant wo man and her fetus, only 20% of women quit smoking duringpregnancy (Hamilton et al. (2004) Natl. Vital Stat. Rep. 53: 1-17). Tostop smoking altogether before or during pregnancy, although ideal, isnot a realistic goal, and therefore any intervention that can safelyprevent or reverse the smoke-induced effects could be an effective,practical strategy to prevent the harmful effects of in utero smokeexposure.

Although there are many agents in smoke that may be detrimental to thedeveloping lung, there is clear evidence showing that nicotine directlyaffects fetal lung development. Nicotine crosses the human placenta withminimal biotransformation to its metabolite cotinine (Pastrakuljic etal. (1998) Life Sci. 63: 2333-2342). In fact, nicotine accumulates infetal blood and amniotic fluid, resulting in the fetus being exposed toeven higher nicotine levels compared to the smoking mother, and itaccumulates in several fetal tissues, including the respiratory tract,suggesting that nicotine is the likely agent that alters lungdevelopmental programming in the fetus of the pregnant smoker (Luck etal. (1985) Dev. Pharmacol. Ther. 8: 3 84-395; Szuts et al. (1978)Toxicology 10: 207-220). The data presented herein suggest thatimprovement in lung development following in utero nicotine exposure canbe accomplished.

Although in utero smoke exposure-induced lung injury is a complexprocess (Pierce and Nguyen (2002) Am. J. Respir. Cell. Mol. Biol. 26:10-13; Rehan et al. (2005) Am. J. Physiol. Lung Cell Mol. Physiol. 289:L667-L676; Proskocil et al. (2005) Am. J. Respir. Crit. Care Med. 171:1032-1039), nicotine-induced LIF-to-MYF transdifferentiation is amechanism that explains many of the pulmonary structural and functionalfindings seen following in utero nicotine exposure. Understanding theprecise molecular mechanism involved in LIF-to-MYF transdifferentiationand its prevention provides a fundamental approach to preventingsmoke-induced lung injury. The hypothesis that PPARγ is a criticaldownstream target for PTHrP/PTHrP receptor signaling and is central tothe lipogenic phenotype of alveolar interstitial fibroblasts (. Rehan etal. (2005) Am. J. Physiol. Lung Cell Mol. Physiol. 289: L667-L676;Torday et al. (2003) Pediatr. Pathol. Mol. Med. 22: 189-207) is yetagain suggested by the data presented in this report: (1) LIF-to-MYFtransdifferentiation was accompanied by PPARy downregulation, (2)reversal of LIF-to-MYF transdifferentiation was accompanied byupregulation of PPARγ, and, most tellingly, (3) the specific PPARγantagonist, GW9662, blocked the reversal of LIF-to-MYFtransdifferentiation by all three upstream stimulators of PPARγ, RGZ,PTHrP, and DBcAMP.

In summary, nicotine-induced LIF-to-MYF transdifferentiation was almostcompletely reversed by treatment with PTHrP, RGZ, or DBcAMP, asdetermined by the expression of the various markers of the LIF and MYFphenotypes. By targeting specific molecular intermediates in the PTHrPsignaling pathway, nicotine-induced LIF-to-MYF transdifferentiation isalmost completely reversible, suggesting not only the possibility ofprevention but also the potential for reversal of nicotine-induced lunginjury.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1-30. (canceled)
 31. A kit for treating a patient at risk for orsuffering from pulmonary damage associated with the transdifferentiationof pulmonary lipofibroblasts to myofibroblasts, the kit comprising: apharmaceutical composition comprising at least one PPARγ agonist in oneor more pharmaceutically-acceptable carrier(s); and a dosage regimenestablishing the amount and frequency of administering thepharmaceutical composition to a patient; wherein the administering atleast one PPARγ agonist prevents, reduces or reversestransdifferentiation of pulmonary lipofibroblasts to myofibroblasts inthe patient when the pharmaceutical composition is administered inaccordance with the dosing regimen.
 32. The kit of claim 31, whereinsaid PPARγ agonist is a thiozolidinedione.
 33. The kit of claim 31,wherein said PPARγ agonist is selected from the group consisting of:rosiglitazone, troglitazone (Resulin), farglitazar, phenylacetic acid,GW590735, GW677954, Avandia, Avandamet (avandia+metformin), ciglitazone,15 deoxy prostaglandin J2 (15PGJ2), pioglitazone (Actos),15-deoxy-deltal-2,14 PGD2, MCC-555, and triterpenoids.
 34. The kit ofclaim 31, wherein the pharmaceutical composition is provided in a dosageform selected from the group consisting of: powders, tablets, pills,capsules, lozenges, suppositories, patches, aerosols, inhalers, nasalsprays, injectibles, implantable sustained-release formulations, andlipid complexes.
 35. The kit of claim 31, wherein the dosing regimenprovides a daily dosing range from about 3 mg/kg to about 100 mg/kg. 36.The kit of claim 35, wherein the daily dosing range is from about 3mg/kg to about 50 mg/kg.
 37. The kit of claim 36, wherein the dailydosing range is from about 3 mg/kg to about 25 mg/kg.
 38. The kit ofclaim 33, wherein the PPARγ agonist is rosiglitazone and wherein thedosing regimen provides a daily dosing range from about 0.1 mg/kg toabout 100 mg/kg.
 39. The kit of claim 38, wherein the daily dosing rangeis from about 1 mg/kg to about 50 mg/kg.
 40. The kit of claim 39,wherein the daily dosing range is from about 3 mg/kg to about 25 mg/kg.41. The kit of claim 31, wherein the patient is diagnosed with one ormore of the following conditions: asthma, chronic obstructive pulmonarydisease, lung cancer or emphysema.
 42. The kit of claim 31, wherein thepharmaceutical composition is contraindicated for patients is notdiagnosed with or being treated for diabetes, obesity, anorexia, aneating disorder or an appetite disorder.
 43. The kit of claim 31,wherein the administering of the PPARγ agonist to the patient preventsthe transdifferentiation of pulmonary lipofibroblasts to myofibroblastsin the patient.
 44. The kit of claim 31, wherein the administering onthe PPARγ agonist to the patient reduces the transdifferentiation ofpulmonary lipofibroblasts to myofibroblasts in the patient.
 45. The kitof claim 31, wherein the administering of the PPARγ agonist to thepatient reverses the transdifferentiation of pulmonary lipofibroblaststo myofibroblasts in the patient.
 46. A method of treating a patient atrisk for or suffering from pulmonary damage associated with thetransdifferentiation of pulmonary lipofibroblasts to myofibroblasts inthe patient, the method comprising: administering a pharmaceuticalcomposition comprising at least one PPARγ agonist to a patient at riskfor or suffering from pulmonary damage associated with thetransdifferentiation of pulmonary lipofibroblasts to myofibroblasts in atherapeutically effective amount over a dosing regimen; wherein theadministering results in the prevention, reduction or reversal oftransdifferentiation of pulmonary lipofibroblasts to myofibroblasts inthe patient.
 47. The method of claim 46, wherein said PPARγ agonist is athiozolidinedione.
 48. The method of claim 46, wherein said PPARγagonist is selected from the group consisting of: rosiglitazone,troglitazone (Resulin), farglitazar, muraglitazar, tesaglitazar,phenylacetic acid, GW590735, GW677954, Avandia, Avandamet(avandia+metformin), ciglitazone, 15 deoxy prostaglandin J2 (15PGJ2),pioglitazone (Actos), 15-deoxy-deltal-2,14 PGD2, MCC-555, andtriterpenoids.
 49. The method of claim 46, wherein said PPARγ agonist isadministered via an inhalation route selected from the group consistingof: oral inhalation and nasal inhalation.
 50. The method of claim 46,wherein said PPARγ agonist is administered orally.
 51. The method ofclaim 46, wherein said PPARγ agonist is administered systemically. 52.The method of claim 46, wherein the patient is diagnosed with one ormore of the following conditions: asthma, chronic obstructive pulmonarydisease, lung cancer or emphysema.
 53. The method of claim 46, whereinthe patient is not diagnosed with or being treated for diabetes,obesity, anorexia, an eating disorder or an appetite disorder.
 54. Themethod of claim 46, wherein the administering results in the preventionof transdifferentiation of pulmonary lipofibroblasts to myofibroblastsin the patient.
 55. The method of claim 46, wherein the administeringresults in the reduction of transdifferentiation of pulmonarylipofibroblasts to myofibroblasts in the patient.
 56. The method ofclaim 46, wherein the administering results in the reversal oftransdifferentiation of pulmonary lipofibroblasts to myofibroblasts inthe patient.